Abstract
Improved methods are described for the detection of G1P-binding proteins (G-proteins) in the protonema of mossFunaria hygrometrica and coleoptiles of corn(Zea mays) and sorghum(Sorghum vulgare). We optimized conditions for the transfer of proteins to nitrocellulose, production of high titer polyclonal anti-Gα (common) antibodies and finally the detection of G-proteins by amplification. In addition to the α-subunit of heterotrimeric G-proteins (M r 41–43 kDa), a small molecular weight class (< 30 kDa) was also detected by anti-Gα (common) antibodies. An easy, reliable and efficient filter assay is also described to quantify the toxin catalyzed ADP-ribosylation. The apparentK m of the NAD has been determined to be approximately 1.5μM for the microsomal fraction of moss. Inclusion of G1P stimulated ADP-ribosylation by 2–27-fold. One to three polypeptides representing the α-subunit of heterotrimeric G-proteins of (Mr 37–43 kDa) were ADP-ribosylated in all three plants. The anti-Gβ (C-terminus) antibody cross-reacted strongly with 39 and 34 kDa polypeptide in moss and corn respectively. By employing improved methods two classes of G-proteins have been shown to be present in three plant species.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bilushi S V, Shebunin A G and Barbakov A V 1991 Purification and subunit composition of a GTP-binding protein from maize root plasma-membranes;FEBS Lett. 291 219–221
Bradford M M 1976 A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing the principle of protein dye binding;Anal. Biochem. 72 248–254
Clarkson J, Finah P M, Ricart C A, White I R and Millner P A 1991 Specific immunodetection of G-proteins in plant cell membranes;Biochem. Soc. Trans. 19 239S.
Dcng X W, Matsui M, Wci N, Wagner D, Chu A M, Feldmann K A and Quail P 1992COPJ. anArabidopsis regulatory gene, encodes a protein with both a zinc-binding motif and aGp homology domain;Cell 71 791–801
Didsbury J, Weber R F, Bokoch G M, Evans T and Synderman R 1989 A novel Ras related famiIy of proteins that are botulinum toxin substrates;J. Biol.Chem. 264 16378–16382
Drobak B K, Allan E F, Comerford J G, Roberts K and Dawson A P 1988 Presence of guanine nucleotide-binding proteins in plant hypocotyl microsomal fraction:Biochem. Biophy. Res. Commun. 150 899–903
Gill D M and Woolkalis M J 1991 Cholera toxin catalyzed [32p] ADP-ribosylation of proteins;Methods Enzymol. 195 267–280
Gilman A G 1987 G proteins: transducers of receptor generated signals;Annu. Rev. Biochem. 6 615–649
Green N, Alexander H, Olson A, Alexander S, Shinnick T M, Sutcliffe J G and Learner R A 1982 Immunogenic structure of Influenza virus hemagglutinin;Cell 28 477–487
Ischda S, Takahashi Y and Nagata T 1993 Isolation of cDNA of an auxin-regulated gene encoding a G-protein subunit-like protein from tobacco BY2 cells;Proc. Natl. Acad. Sci. USA 90 11152–11156
Johri M M 1974 Differentiation of caulonema cells by auxins in suspension cultures ofFunaria hygrometrica; inPlant growth substances 1973 (Tokyo: Hirokawa Publishing Co) pp 925–933
Kaziro Y, Itoh H, Kozasa T, Nakafuka M and Satoh T 1991 Structure and function of signal transducing GTP-binding proteins;Annu. Rev. Biochem. 60 349–400
Kitajima S, Seto-Ohshima A, Sano M and Kato K 1983 Production of antibodies to calmodulin in rabbits and enzyme immuno-assays for calmodulin and anti-calmodulin;J. Biochem. 94 559–564
Laemmli U K 1970 Cleavage of structural proteins during the assembly of head bacteriophage T4;Nature (London) 227 680–685
Ma H 1994 GTP-binding proteins in plants: new members of an old family;Plant. Mol. Bioi. 26 1611–1636
Mitchell F M, Griffiths S L, Saggerson E D, Houslay M D, Knowler J T and Milligan G 1989 Guanine-nucleotide-binding proteins expressed in rat white adipose tissue;Biochem. J. 262 403–408
Mumby S M and Gilman A G 1991 Synthetic peptide antisera with determined specificity for G-protein α or β-subunits;Methods. Enzymol. 195 215–233
Perdue D O and Hurley J B 1993 Identification and partial purification of GTP-binding proteins and nucleotide diphosphate kinase from membranes of Zucchini;Plant Physiol. Suppl. 102 13
Ricart C A O. White I R, Findlay J B C, Keen J N and Millner P A 1995 Detection and purification of a putative Gao protein fromSorghum bicolor;J. Plant Physiol. 146 645–651
Schloss J A 1990 AChlamydamollllS gene encodes a G-protein J-subunit-like polypeptide;Mol. Gen. Genet. 221 443–452
Sekine A, Fujiwara M and Narumiya S 1989 Asparagine residue in the rho gene product is the modification substrate for botulinum ADP ribosyl-transferase;J. Biol. Chem. 264 8602–8605
Simon M I, Strathmann M P and Gautam N 1991 Diversity of G-proteins in signal transduction;Science 252 802–808
Thelu J, Bracchi V, Burnod J and Ambroise-Thomas P 1994 Evidence for Ras-like and a stage specific GTP binding homologous protein byPlasmodium falciparum;Cell Signal 6 777–782
Towbin H, Staelehin T and Gordon J 1989 Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets;Proc. Natl. Acad. Sci. USA 76 4350–4354
Weiss C A, Gamatt C W, Mukai K, Hu Y and Ma H 1994 Isolation of cDNAs encoding G-proteinβ subunit homologues from maize (ZMB 1) andArabidopsis (AGB1);Proc. Natl. Acad. Sci. USA 91 9554–9558
Zaina S, Breviario D, Mapelli S, Bertini A, Reggiani R 1994 Two putative G-protein a-subunits dissociate from rice coleoptile membranes after GTP stimulation;J. Plant Physiol. 143 293–297
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Panigrahi, K.C.S., Johri, M.M. Improved methods to detect GTP-binding proteins from plants. J. Biosci. 23, 193–200 (1998). https://doi.org/10.1007/BF02720020
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/BF02720020