Antisera against the 3-17 sequence of rat G alpha i recognize only a 40 kDa G-protein in brain

To obtain antisera specific for the GTP-binding protein Gi alpha we immunized rabbits against a synthetic peptide derived from the N-terminal (3-17) sequence predicted from the rat Gi alpha cDNA clone published by Itoh et al. (1986) (Proc. Natl. Acad. Sci. USA 83, 3776-3780). Western-blot analysis of bovine brain G-proteins purified and resolved by hydrophobic chromatography and of rat striatal membranes, indicate that this antiserum does not recognize 41 kDa alpha i or 39 kDa alpha o. However, it reacts with a 40 kDa alpha-subunit. The data suggest that the sequence deduced from the rat G alpha i cDNA corresponds to a G40 alpha protein and that N-terminus directed antisera are useful tools to discriminate between two different G alpha i-like types of G-proteins present in mammalian brain.     

Chromatographic resolution and immunologic identification of the alpha 40 and alpha 41 subunits of guanine nucleotide-binding regulatory proteins from bovine brain

A guanine nucleotide-binding regulatory protein (G protein), with subunits designated as alpha 40 beta gamma, was identified and partially resolved from two other purified G proteins, Go (alpha 39 beta gamma) and Gi (alpha 41 beta gamma), found in bovine brain. The alpha 40 G protein subunit served as a substrate for ADP-ribosylation catalyzed by Bordetella pertussis toxin, as did alpha 39 and alpha 41. alpha 40 was shown to be closely related to, but distinct from, alpha 41 by reaction with various peptide antisera. An antiserum generated against a peptide derived from the sequence of a Gi alpha clone isolated from a rat C6 glioma cDNA library (Itoh, H., Kozasa, T., Nagata, S., Nakamura, S., Katada, T., Ui, M., Iwai, S., Ohtsuka, E., Kawasaki, H., Suzuki, K., and Kaziro, Y. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 3776-3780) reacted with alpha 40 to the exclusion of all other alpha subunits tested. Another antiserum generated against a peptide derived from an analogous region of a different Gi alpha clone from a bovine brain cDNA library (Nukuda, T., Tanabe, T., Takahashi, H., Noda, M., Haga, K., Haga, T., Ichiyama, A., Kangawa, K., Hiranaga, M., Matsuo, H., and Numa, S. (1986) FEBS Lett. 197, 305-310) reacted exclusively with alpha 41. Evidence is given for the existence of another form of alpha 41 that did not react with either of these two peptide antisera. The antisera were used to survey various rat tissues for the expression of alpha 40 and alpha 41.     

A 43 kDa form of the GTP-binding protein Gi3 in human erythrocytes

Purified preparations of human erythrocyte G-proteins contain a 43 kDa pertussis toxin substrate which appears to be the alpha-subunit of a heterotrimeric GTP-binding protein. The 43 kDa protein is recognized by antisera that are sequence-specific for peptides encoding a sequence common to all 39-53 kDa G-protein alpha-subunits. G alpha o-specific antiserum did not recognize 43 or 40-41 kDa alpha-subunits. AS/6, which recognizes the alpha i proteins, recognized 43 kDa as well as 40-41 kDa proteins. Of the three antisera specific for individual members of the alpha i family, only the Gi3-specific antiserum recognized the 43 kDa erythrocyte G-protein. However, 40-41 kDa forms of all three alpha is are present. These observations indicate that human erythrocytes contain a novel 43 kDa form of Gi3.     

Identification of a new GTP-binding protein. A Mr = 43,000 substrate for pertussis toxin

In purified preparations of human erythrocyte GTP-binding proteins, we have identified a new substrate for pertussis toxin, which has an apparent molecular mass of 43 kDa by silver and Coomassie Blue staining. Pertussis toxin-catalyzed ADP-ribosylation of the 43-kDa protein is inhibited by Mg2+ ion and this inhibition is relieved by the co-addition of micromolar amounts of guanine nucleotides. GTP affects the ADP-ribosylation with a K value of 0.8 microM. Addition of a 10-fold molar excess of purified beta gamma subunits (Mr = 35,000 beta; and Mr = 7,000 gamma) of other GTP-binding proteins results in a significant decrease in the pertussis toxin-mediated ADP-ribosylation of the 43-kDa protein. Treatment of the GTP-binding proteins with guanosine 5'-O-(thiotriphosphate) and 50 mM MgCl2 resulted in shifting of the 43-kDa protein from 4 S to 2 S on sucrose density gradients. Immunoblotting analysis of the 43-kDa protein with the antiserum A-569, raised against a peptide whose sequence is found in the alpha subunits of all of the known GTP-binding, signal-transducing proteins (Mumby, S. M., Kahn, R. A., Manning, D. R., and Gilman, A. G. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 265-259) showed that the 43-kDa protein is specifically recognized by the common peptide antiserum. A pertussis toxin substrate of similar molecular weight was observed in human erythrocyte membranes, bovine brain membranes, membranes made from the pituitary cell line GH4C1, in partially purified GTP-binding protein preparations of rat liver, and in human neutrophil membranes. Treatment of neutrophils with pertussis toxin prior to preparation of the membranes resulted in abolishment of the radiolabeling of this protein. From these data, we conclude that we have found a new pertussis toxin substrate that is a likely GTP-binding protein.     

Purification of an AlF4- and G-protein beta gamma-subunit-regulated phospholipase C-activating protein.

A 150-kDa phospholipase C has previously been purified from turkey erythrocytes and has been shown by reconstitution with turkey erythrocyte membranes to be a receptor- and G-protein-regulated enzyme (Morris, A. J., Waldo, G. L., Downes, C.P., and Harden, T. K. (1990) J. Biol. Chem. 265, 13501-13507; Morris, A.J., Waldo, G.L., Downes, C.P., and Harden, T.K. (1990) J. Biol. Chem. 265, 13508-13514). Combination of this 150-kDa protein with phosphoinositide substrate-containing phospholipid vesicles prepared with a cholate extract from purified turkey erythrocyte plasma membranes resulted in conferrence of AlF4- sensitivity to the purified phospholipase C. Guanosine 5'-3-O-(thio)triphosphate also activated the reconstituted phospholipase C in a manner that was inhibited by guanosine 5'-2-O-(thio)-diphosphate. The magnitude of the AlF4- stimulation was increased with increasing amounts of plasma membrane extract, and was also dependent on the concentration of purified phospholipase C. Using reconstitution of AlF4- sensitivity as an assay, the putative G-protein conferring regulation to the 150-kDa phospholipase C was purified to near homogeneity by sequential chromatography over Q-Sepharose, Sephacryl S-300, octyl-Sepharose, hydroxylapatite, and Mono-Q. Reconstituting activity co-purified with an approximately 43-kDa protein identified by silver staining; lesser amounts of a 35-kDa protein was present in the final purified fractions, as was a minor 40-kDa protein. The 43-kDa protein strongly reacted with antiserum against a 12-amino acid sequence found at the carboxyl terminus of Gq and G11, the 35-kDa protein strongly reacted with G-protein beta-subunit antiserum, and the 40-kDa protein reacted with antiserum that recognizes Gi3. Immunoprecipitation of the 43-kDa protein resulted in loss of phospholipase C-stimulating activity of the purified fraction. The idea that this is a phospholipase C-regulating G-protein is further supported by the observation that co-reconstitution of G-protein beta gamma-subunit with the purified phospholipase C-activating fraction resulted in a beta gamma-subunit-dependent inhibition of AlF(4-)-stimulated phospholipase C activity in the reconstituted preparation.     

Immunochemical detection of the alpha-subunit of the G-protein, GZ, in membranes and cytosols of mammalian cells

An antiserum (AS 98) was raised against a synthetic peptide deduced from published cDNA sequences of the alpha-subunit of the putative G-protein, GZ (Fong et al. Proc. Natl. Acad. Sci. USA 85, 3066-3070, 1988; Matsuoka et al. Proc. Natl. Acad. Sci. USA 85, 5384-5388, 1988). In membrane and cytosol preparations of many but not all tested mammalian tissues, AS 98 predominantly recognized two proteins of 40 and 43 kDa Mr. Whereas high levels of a 40 kDa GZ alpha-subunit were found in rat liver membranes and in brain cytosol, AS 98 failed to detect the alpha-subunit of GZ in brain membranes.     

Identification of the GTP-binding protein encoded by Gi3 complementary DNA

Three closely related, but distinct, GTP-binding proteins (G-proteins) are encoded by cDNAs arbitrarily designated Gi1, Gi2, and Gi3. The in vitro translated products of mRNAs prepared from Gi1, Gi2, and Gi3 cDNAs migrate as 41-, 40-, and 41-kDa proteins, respectively, on sodium dodecyl sulfate-polyacrylamide gels. Antisera were raised against synthetic decapeptides corresponding to a divergent sequence (residues 159-168 for Gi1 and Gi3; 160-169 for Gi2) of the three cDNAs and tested on immunoblots for reactivity with three purified G-proteins, G41 and G40 from brain and G41 from HL-60 cells. LD antisera (Gi1 peptide) react only with brain G41. LE antisera (Gi2 peptide) react only with brain G40, and SQ antisera (Gi3 peptide) react exclusively with HL-60 G41. The results indicate that the 41-kDa G-protein purified from HL-60 cells differs from the purified brain 41-kDa protein and suggest that the HL-60 cell protein corresponds to that encoded by Gi3 cDNA.     

Purification of p100, a protein antigenically related to the signal transducing G proteins Gt and Gi. Evidence for an adaptin-like protein.

A 100-kDa protein, termed p100, cross-reacts with antisera raised against a synthetic peptide corresponding to the carboxyl-terminal decapeptide of the alpha-subunit of the retinal G protein Gt. p100 is abundantly expressed in liver and, on subcellular fractionation of rat liver homogenates, is distributed between the cytosolic and microsome fractions (Traub, L. M., Evans, W. H., and Sagi-Eisenberg, R. (1990) Biochem. J. 272, 453-458; Udrisar, D., and Rodbell, M. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 6321-6325). We have now purified p100 to near-homogeneity from rat liver microsomes. The protein was purified approximately 500-fold by ATP extraction followed by a series of four chromatographic steps. Similar to partially purified p100, on two-dimensional electrophoresis, the final preparation contained a major series of five immunoreactive 100-kDa charge isoforms. Partial amino terminus amino acid sequencing of the purified protein revealed that p100 is a previously unidentified protein. Further analysis of the soluble form of p100 showed the protein migrated with an apparent molecular weight of approximately 110,000 on gel filtration, indicating that the soluble protein occurs as a monomeric polypeptide. The soluble form of p100 was also partially purified from rat liver cytosol and amino acid sequencing yielded the same amino-terminal sequence as obtained from the microsome-associated form. The amino-terminal sequence of p100 exhibits significant similarity to the deduced amino-terminal amino acid sequences of both alpha- and gamma-adaptins. Using the amino-terminal sequence from p100, we have raised antipeptide polyclonal antisera. The antisera reacted specifically with the purified 100-kDa protein on immunoblots. With the purified protein and specific antisera now available, it will be possible to explore the physiological role of p100.     

Structural analysis of the sequence coding for an inducible 26-kDa protein in human fibroblasts

When human fibroblast cells were stimulated with poly(I) X poly(C) in the presence of cycloheximide for the production of interferon-beta (IFN-beta), a 26-kDa protein could be immunoprecipitated by antiserum raised against partially purified human IFN-beta [Content, J., De Wit, L., Pierard, D., Derynck, R., De Clercq, E. & Fiers, W. (1982) Proc. Natl Acad. Sci. USA 79, 2768-2772]. In our hands this 26-kDa protein showed no antiviral activity. Other investigators have, however, reported the presence in the same conditions of a second type of IFN, a so-called beta 2 species [Weissenbach, J., Chernajovsky, Y., Zeevi, M., Shulman, L., Soreq, H., Nir, U., Wallach, D., Perricaudet, M., Tiollais, P. & Revel, M. (1980) Proc. Natl Acad. Sci. USA 77, 7152-7156] of which the mRNA structure and protein characteristics strongly suggests identity with the 26-kDa product. In this paper we describe the nucleotide sequence of the 26-kDa cDNA and part of the corresponding genomic clone. The cDNA clones were isolated from a library made with mRNA from induced human fibroblasts. As, however, the information thus obtained was still incomplete, genomic clones were isolated from a total human DNA library. In this way, the entire region coding for the 26-kDa protein was established, as well as the neighbouring sequences including the inducible promoter area. From the deduced polypeptide sequence a number of characteristics of the 26-kDa protein can be explained. It turns out that the 26-kDa protein gene and the so-called 'IFN-beta 2' gene are identical. However, extensive homology searches indicate that the 26-kDa protein does not show statistically significant sequence homology with any known interferon species. Hence, the question of whether the 26-kDa product represents a novel IFN species remains open.     

Antibodies directed against synthetic peptides distinguish between GTP-binding proteins in neutrophil and brain

Antisera AS/6 and 7, raised against a synthetic peptide KENLKDCGLF corresponding to the carboxyl-terminal decapeptide of transducin-alpha, react on immunoblots with purified transducin-alpha and with proteins of 40-41 kDa in all tissues tested. The latter represent one or more forms of Gi alpha but not Go alpha, since a synthetic peptide, KNNLKDCGLF, corresponding to the carboxyl-terminal decapeptide of two forms of Gi alpha blocks AS/6 and 7 reactivity with transducin-alpha and Gi alpha on immunoblots, whereas the corresponding Go-related peptide, ANNLRGCGLY, does not. Antisera LE/2 and 3, raised against the synthetic peptide LERIAQSDYI, corresponding to an internal sequence predicted by one form of Gi alpha cDNA (Gi alpha-2) and differing by 3 residues from the sequence of another form, Gi alpha-1, react strongly with a 40-kDa protein abundant in neutrophil membranes and with the major pertussis toxin substrate purified from bovine neutrophils. LE/2 and 3 reveal a relatively faint 40-kDa band on immunoblots of crude brain membranes or of purified brain Gi/Go. LE/2 and 3 do not react with transducin-alpha or Go alpha nor with the 41-kDa form of pertussis toxin substrate in brain, Gi alpha-1. These antisera distinguish between the major pertussis toxin substrates of brain and neutrophil and tentatively identify the latter as Gi alpha-2.     

Purification of unique alpha subunits of GTP-binding regulatory proteins (G proteins) by affinity chromatography with immobilized beta gamma subunits

Novel G protein alpha subunits were purified from rat brain by an affinity matrix containing immobilized beta gamma subunits (Pang, I.-H., and Sternweis, P. C. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 7814-7818). They were unique based on the following criteria. These alpha subunits migrated differently through polyacrylamide gels with an apparent molecular mass of 42 kDa. They did not behave similarly to the other brain G proteins by conventional chromatographic techniques. Antisera raised against a common region of known alpha subunits failed to recognize these 42-kDa polypeptides. Finally, primary sequences of tryptic fragments of these proteins contain regions homologous to, yet unique from, the other alpha subunits. Sequences are identical with one or more members of a new family of alpha subunits recently identified by molecular genetic techniques (Strathmann, M., Wilke, T. M., and Simon, M. I. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 7407-7409); most of the primary sequence identifies an alpha subunit labeled alpha q. These polypeptides were not substrates for ADP-ribosylation catalyzed by pertussis toxin. They bound GTP gamma S only with slow rates and low stoichiometry. Antisera to peptides based on primary sequence were specific for the new alpha subunits and indicate that they are widely distributed at low levels in different tissues but more concentrated in brain and lung. This procedure provides a means of preparing native G proteins that have a potential role as modulators of pertussis toxin-insensitive regulatory pathways.     

Characterization of a human recombinant receptor-linked protein tyrosine phosphatase.

The receptor-linked tyrosine phosphatase RPTP alpha from human brain (Kaplan, R., Morse, B., Huebner, K., Croce, C., Howk, R., Ravera, M., Ricca, G., Jaye, M., and Schlessinger, J. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7000-7004) was expressed in insect cells following infection with recombinant baculovirus. Two major forms of the enzyme, with molecular sizes of 98 kDa and 114 kDa, were detected by immunoblot analysis. This heterogeneity could be ascribed to N-linked glycosylation on the basis of two lines of evidence; namely, blockage of glycosylation with tunicamycin in vivo and removal of carbohydrates by endoglycosidase F in vitro. The 114-kDa form was purified to homogeneity by chromatography on Superose 12 and Mono Q. Compared to the low Mr placenta and T-cell tyrosine phosphatases, RPTP alpha displayed a low optimum pH of 6 and a high Km in the micromolar range toward two artificial substrates (tyrosyl-phosphorylated myelin basic protein and modified lysozyme, respectively). Most effectors had a different and often an opposite influence on phosphatase activity depending on the nature of the substrate and the pH at which the assays were performed. Determination of Km and Vmax values for RPTP alpha suggests that the enzyme could exist in low and high substrate affinity states.     

A gene encoding the proteolipid subunit of Sulfolobus acidocaldarius ATPase complex

An analysis of genes for the major two subunits of the membrane-associated ATPase from an acidothermophilic archaebacterium, Sulfolobus acidocaldarius, suggested that it belongs to a different ATPase family from the F1-ATPase (Denda, K., Konishi, J., Oshima, T., Date, T., and Yoshida, M. (1988) J. Biol. Chem. 263, 17251-17254). In the same operon of the above two genes we found a gene encoding a very hydrophobic protein of 101 amino acids (Mr = 10,362). A proteolipid was purified from the membranes of this bacteria in which partial amino acid sequences matched with the sequence deduced from the gene. Significant amino acid sequence homology and a similar hydropathy profile appeared when the sequence was compared with the 8-kDa proteolipid subunit of F0F1-ATPases. It is about 30 amino acids larger than the 8-kDa proteolipid and has a small (11-amino acid) repeat sequence. However, it is distinct from the 16-kDa proteolipid subunit of an eukaryotic vacuolar H+-ATPase (Mandel, M., Moriyama, Y., Hulmes, J.D., Pan, Y.-E., Nelson, H., and Nelson, N. (1988) Proc. Natl. Acad. Sci. U.S.A. 85,5521-5524).     

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.     

Native species of helix destabilizing protein-1 in mouse myeloma identified by antibody probing of Western blots

Antibody probing of Western blots is a method for analyzing the apparent Mr of a protein in any given preparation (Renart, J., Reiser, J., and Stark, G., Proc. Natl. Acad. Sci. USA 76: 3116, 1979). We prepared a rabbit antiserum to purified mouse myeloma helix destabilizing protein-1 and used this antiserum in Western blotting experiments with a crude homogenate of mouse myeloma. The results indicated that the native species of helix destabilizing protein-1 can be degraded during purification. This in vitro proteolysis results in complete loss of the native species and accumulation of lower Mr proteins that represent limit digestion products. These findings have identified the true native form of mouse myeloma HDP-1 as a protein of apparent Mr = 36,000 to 38,000, instead of the Mr = 24,000 and 27,000 proteins obtained by routine purification.     

Complexation of fibronectin with tissue transglutaminase

Previous work [Lorand, L., Dailey, J. E., & Turner, P. M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 1057-1059] showed that fibronectin might serve as a specific carrier for transglutaminases accidentally discharged from erythrocytes or other cells into plasma. In the present study we examined the association of these proteins in purified systems. Complexation was readily demonstrable by nondenaturing electrophoresis, using dansylcadaverine-dependent activity staining as well as immunoblotting procedures, and also by HPLC gel filtration. The results indicate a stoichiometry of 2:1 for the binding of the human erythrocyte transglutaminase (80K) to human plasma fibronectin (440K). The attachment is noncovalent in nature and does not involve cross-linking of the proteins either to themselves or to each other. Binding occurs in the absence of Ca2+, suggesting that a domain on the transglutaminase molecule other than the catalytic site is needed for complexation with fibronectin. Limited proteolysis with chymotrypsin for delineating the relevant region in fibronectin yielded two gelatin- (collagen) binding fragments (56K and 46K), each displaying affinity for transglutaminase. Moreover, these fragments--like intact fibronectin--bound erythrocyte transglutaminase and gelatin simultaneously in ternary complexes.     

Immunological identification of G protein alpha- and beta-subunits in tail membranes of bovine spermatozoa.

Heterotrimeric G proteins are believed to play important roles as signal transducing components in various mammalian sperm functions. To assess the distribution of G proteins in bovine sperm tails, we purified membranes by hypoosmotic swelling of bovine spermatozoa followed by disruption of plasma membranes in a homogenizer and various centrifugation steps. Electron microscopy revealed highly purified membranes of bovine sperm tails. Subsequently, antisera against synthetic peptides were used to identify G proteins in immunoblots. An antiserum directed against the C-terminal decapeptide of Gi3 and detecting all known pertussis toxin-sensitive alpha-subunits, reacted specifically with a 40-kDa protein. In contrast, various other specific peptide antisera against alpha-subunits did not detect any G protein in enriched tail membranes. An antiserum recognizing the beta 2-subunit of G proteins and an antiserum reacting with both beta 1- and beta 2-subunits identified a 35-kDa protein in sperm tail membranes. In contrast, antisera against the 36-kDa beta 1-subunit did not detect any relevant proteins in the membrane fraction. Neither G protein alpha-subunits nor G protein beta-subunits were found in the cytosol. Our results suggest that G proteins in membranes of tails of bovine spermatozoa most likely belong to a novel subtype of G protein alpha-subunits, whereas the putative beta-subunit could be identified as a beta 2-subunit.