共查询到20条相似文献,搜索用时 62 毫秒
1.
Molecular cloning of a new human G protein. Evidence for two Gi alpha-like protein families 总被引:9,自引:0,他引:9
The amino acid sequence of a novel G protein alpha subunit (Gx alpha) has been deduced from the nucleotide sequence of a human cDNA clone isolated from a differentiated HL-60 cDNA library. The cDNA encodes a polypeptide of 354 amino acids (Mr 40,519) which is closely related to Gi alpha proteins. The amino acid sequence homology between Gx alpha and human myeloid Gi alpha is 86% with 15 nonconservative substitutions. Gx alpha also shares 86% homology with both rat brain and mouse macrophage Gi alpha but is more homologous (94%) to bovine brain Gi alpha with only 5 nonconservative amino acid differences. G proteins previously termed Gi alpha may fall into at least two distinct groups, with one including human myeloid Gi alpha, rat brain Gi alpha and mouse macrophage Gi alpha; and other Gx alpha and bovine brain Gi alpha. One group probably contains true Gi and the other a new class of G protein whose function remains to be determined. 相似文献
2.
Go, a major brain GTP binding protein in search of a function: purification, immunological and biochemical characteristics 总被引:4,自引:0,他引:4
The GTP-binding proteins involved in signal transduction now constitute a large family of so called 'G proteins'. Among them, Gs and Gi mediate the stimulation and inhibition of adenyl cyclase, respectively. Recently, another G protein (Go) abundant in brain was purified, but its function is still unknown. Like other G proteins, Go is a heterotrimer (alpha, beta, gamma) and the beta-gamma subunits seem to be identical to those of Gs and Gi. The alpha subunit of Go (Go-alpha) has a molecular weight of 39 kDa lower than those of Gi (41 kDa) or Gs (45-52 kDa). A positive immunoreativity with antibodies against Go-alpha was found in peripheral nervous tissues, adrenal medulla, heart, adenohypophysis and adipocytes. Go ressembles Gi in its ability to be ADP-ribosylated by pertussis toxin, and sequence analysis reveals a 68% homology between their alpha subunits. The GTPase activity of Go is several times higher than that of Gi. The affinity of the beta-gamma entity is about 3 times higher for Gi than for Go. In reconstitution studies, Go does not mimic the inhibitory effect of Gi on adenyl cyclase-stimulated by Gs. On the contrary, Go is as efficient as Gi in reconstituting the functional coupling with the muscarinic, alpha 2-adrenergic and chemotactic agent f-Met-Leu-Phe (fMLP), receptors. Recent studies seem to rule out Go as the coupling G protein of phospholipase C, the enzyme involved in phosphatidyl inositol trisphosphate hydrolysis. However, Go remains a putative candidate for transduction mechanisms coupled to a potassium channel or to a voltage-dependent calcium channel. 相似文献
3.
Amino acid sequence determination of the novel forms of Go alpha purified from bovine brain membranes 总被引:1,自引:0,他引:1
Previously we have reported that there are at least four different forms of Go alpha in bovine brain membranes which can be distinguished by their elution profiles from Mono Q column and their immunological reactivities. The four alpha-subunits are referred to as alpha o1, alpha o2, alpha o3 and alpha o4 in their elution orders from the column. Partial amino acid sequences of the purified alpha o1 and alpha o2 were determined and compared with the predicted sequences of two classes of Go alpha cDNAs, termed Go alpha-1 and Go alpha-2. There were at least two unique fragments corresponding with the predicted amino acid sequence of the Go alpha-2 cDNA but different from that of the Go alpha-1 cDNA upon tryptic digestion of alpha o1- or alpha o2-subunit. The alpha o3- and alpha o4-subunits, but not alpha o1-and alpha o2-subunits, were recognized by an antibody raised against a unique amino acid sequence predicted from Go alpha-1 cDNA. These results suggest that alpha o1,2 subunits and alpha o3,4 subunits are encoded by Go alpha-2 cDNA and Go alpha-1 cDNA, respectively. 相似文献
4.
We have determined the partial amino acid sequences of the 40 kDa protein, one of the three pertussis toxin substrates in porcine brain. Purified 40 kDa protein from porcine brain was completely digested with TPCK-trypsin. Digested peptides were separated by reverse-phase HPLC and subjected to analysis by gas-phase protein sequencing. Several sequences of porcine brain 40 kDa protein completely matched with those which were deduced from the nucleotide sequences of the human Gi2 alpha gene and rat Gi2 alpha cDNA. On the other hand, the previously determined sequences of the rat brain 41 and 39 kDa proteins were in complete agreement with the predicted amino acid sequences of rat Gi1 alpha and Go alpha cDNAs, respectively. 相似文献
5.
J J Cali E A Balcueva I Rybalkin J D Robishaw 《The Journal of biological chemistry》1992,267(33):24023-24027
The GTP-binding regulatory proteins (G proteins) that transduce signals from receptors to effectors are composed of alpha, beta, and gamma subunits. Whereas the role of alpha subunits in directly regulating effector activity is widely accepted, it has recently been demonstrated that beta gamma subunits may also directly regulate effector activity. This has made clear the importance of identifying and characterizing beta and gamma subunits. We have isolated a cDNA clone encoding a new gamma subunit, referred to here as the gamma 7 subunit, using probes based on peptide sequences of a gamma subunit previously purified from bovine brain. The clone contains a 1.47-kilobase cDNA insert, which includes an open reading frame of 204 base pairs that predicts a 68-amino acid polypeptide with a calculated M(r) of 7553. The predicted protein shares amino acid identities with the other known gamma subunits, ranging from 38 to 68%. Also characteristic of gamma subunits is a carboxyl-terminal CAAX motif. The expression of the gamma 7 subunit as well as the gamma 2, gamma 3, and gamma 5 subunits was examined in several bovine tissues at both the mRNA and protein levels. Whereas the gamma 2 and gamma 3 subunits were selectively expressed in brain, the gamma 5 and gamma 7 subunits were expressed in a variety of tissues. Thus, the gamma 5 and gamma 7 subunits are the first G protein gamma subunits known that could participate in the regulation of widely distributed signal transduction pathways. 相似文献
6.
Peptide alignment of the porcine 21-hydroxylase cytochrome P-450 using a cDNA sequence of the corresponding bovine enzyme 总被引:2,自引:0,他引:2
M J Bienkowski M Haniu S Nakajin M Shinoda K Yanagibashi P F Hall J E Shively 《Biochemical and biophysical research communications》1984,125(2):734-740
The amino acid sequence deduced from a cDNA clone of the bovine adrenal steroid 21-hydroxylase cytochrome P-450 has been utilized to align peptide sequences derived from the corresponding porcine enzyme. Homology analysis revealed that only fifty percent of the amino acid sequence predicted by the cDNA clone overlapped with peptide sequences from the porcine enzyme. The homology in the remaining portions of the bovine sequence was restored by considering amino acid sequences predicted by the two additional DNA reading frames of the cDNA sequence. Forty eight percent of the bovine sequences predicted by the two alternate reading frames showed strong homology with the porcine peptide sequences. A minimum of 4 nucleotide sequencing errors account for the observed reading frame alterations and the approximate position of each error in the bovine cDNA sequence has been established. 相似文献
7.
Sequence analysis of the catalytic subunit of H(+)-ATPase from porcine renal brush-border membranes.
I Sander F Lottspeich H Appelhans E Kojro J Spangenberg C Weindel W Haase H Koepsell 《Biochimica et biophysica acta》1992,1112(1):129-141
The catalytic subunit of the H(+)-ATPase from brush-border membranes of porcine renal proximal tubules was labeled with the hydrophobic SH-group reagent 10-N-(bromoacetyl)amino-1-decyl-beta-glucopyranoside (BADG) which irreversibly inhibits proton pump activity in the absence but not in the presence of ATP. The labeled protein was purified and digested with proteinases. After isolation and sequencing of proteolytic peptides two BADG-labeled cysteines were identified. The amino acid sequences of the obtained proteolytic peptides were homologous to the catalytic subunit of V-ATPases. From mRNA of porcine kidney cortex a catalytic H(+)-ATPase subunit was cloned. 181 of the 183 amino acids which overlap in the sequence derived from the cDNA and the proteolytic peptides were identical, and the two deviations are due to single base exchanges. A comparison of the amino acid sequence derived from the cloned cDNA with sequences of catalytic H(+)-ATPase subunits communicated by other laboratories revealed 98%, 96% and 94% identity with sequences from bovine adrenal medulla, from bovine kidney medulla and from clathrin-coated vesicles of bovine brain. Between 64% and 69% identity was obtained with sequences from fungi and plants. The data show that the catalytic subunit of V-ATPases is highly conserved during evolution. They indicate organ and species specificity in mammalians. 相似文献
8.
Molecular cloning of cDNA encoding the C subunit of H(+)-ATPase from bovine chromaffin granules 总被引:1,自引:0,他引:1
H Nelson S Mandiyan T Noumi Y Moriyama M C Miedel N Nelson 《The Journal of biological chemistry》1990,265(33):20390-20393
A cDNA encoding subunit C of the V-ATPase from bovine chromaffin granules was cloned and sequenced. The gene encodes a hydrophilic protein of 382 amino acids with a calculated molecular weight of 43,989. Hydropathy plots revealed no apparent transmembrane segments and a rather high helix content was detected. A cDNA encoding most of the C subunit of the V-ATPase of human brain was also cloned and sequenced. The deduced amino acid sequence of this gene is almost identical to the bovine polypeptide with only one change of tyrosine 336 that was replaced by histidine in the human gene. Two polypeptide fragments derived from subunit E of V-ATPase from chromaffin granules were sequenced and found to be identical to the predicted amino acid sequence of this subunit from bovine kidney. These observations support the idea that the amino acid sequences of corresponding subunits from different V-ATPases are highly conserved. Unlike the A and B subunits of V-ATPases, that are homologous to the beta and alpha subunits of F-ATPases, subunits C and E showed no homology with analogous subunits of the F-ATPase family. It is proposed that the addition of the C and gamma subunits to the respective V- and F-ATPases during evolution defined them as two separate families of H(+)-ATPases. 相似文献
9.
Major pertussis-toxin-sensitive GTP-binding protein of bovine lung. Purification, characterization and production of specific antibodies 总被引:2,自引:0,他引:2
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. 相似文献
10.
We have examined the ability of the beta gamma subunits of guanine nucleotide binding regulatory proteins (G proteins) to support the pertussis toxin (PT) catalyzed ADP-ribosylation of G protein alpha subunits. Substoichiometric amounts of the beta gamma complex purified from either bovine brain G proteins or the bovine retinal G protein, Gt, are sufficient to support the ADP-ribosylation of the alpha subunits of Gi (the G protein that mediates inhibition of adenylyl cyclase) and Go (a G protein of unknown function) by PT. This observation indicates that ADP-ribosylated G protein oligomers can dissociate into their respective alpha and beta gamma subunits in the absence of activating regulatory ligands, i.e., nonhydrolyzable GTP analogues or fluoride. Additionally, the catalytic support of ADP-ribosylation by bovine brain beta gamma does not require Mg2+. Although the beta gamma subunit complexes purified from bovine brain G proteins and the beta gamma complex of Gt support equally the ADP-ribosylation of alpha subunits by PT, there is a marked difference in their abilities to interact with Gs alpha. The enhancement of deactivation of fluoride-activated Gs alpha requires 25-fold more beta gamma from Gt than from brain G proteins to produce a similar response. This difference in potency of beta gamma complexes from the two sources was also observed in the ability of beta gamma to produce an increase in the activity of recombinant Gs alpha produced in Escherichia coli. 相似文献
11.
S Mumby I H Pang A G Gilman P C Sternweis 《The Journal of biological chemistry》1988,263(4):2020-2026
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. 相似文献
12.
Expression of Go alpha mRNA and protein in bovine tissues 总被引:4,自引:0,他引:4
S R Price S C Tsai R Adamik C W Angus I M Serventi M Tsuchiya J Moss M Vaughan 《Biochemistry》1989,28(9):3803-3807
Go alpha is a 39-kDa guanine nucleotide-binding protein (G protein) similar in structure and function to Gs alpha and Gi alpha of the adenylate cyclase complex and to transducin (Gt alpha) of the retinal photon receptor system. Although expression of Go alpha protein has been reported to be tissue-specific, other workers have found Go alpha mRNA in all rat tissues examined. In order to clarify this contradiction, studies to verify the distribution of Go alpha mRNA and protein in bovine and rat tissues were performed. Tissues were screened for the presence of Go alpha mRNA by use of a series of restriction fragments of a bovine retinal cDNA clone, lambda GO9, and oligonucleotide probes complementary to sequences specific among G alpha subunits for the 5' untranslated and coding regions of Go alpha. These probes hybridized predominantly with mRNA of 4.0 and 3.0 kb in bovine brain and retina. A 2.0-kb mRNA in retina also hybridized strongly with the cDNA but weakly with the oligonucleotide probes. In bovine lung, two mRNAs of 1.6 and 1.8 kb hybridized with the cDNA while only the 1.6-kb species hybridized with the coding-region oligonucleotide. In bovine heart, only a 4.0-kb mRNA was detected and in amounts much less than those in the other tissues. A similar distribution of Go alpha mRNAs was seen in rat tissues. In bovine tissues, Go alpha protein was identified with rabbit polyclonal antibodies directed against purified bovine brain Go alpha. An immunoreactive 39-kDa membrane protein was found principally in retina and brain, and in a lesser amount in heart.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
13.
The carboxy-terminal domain of Gs alpha is necessary for anchorage of the activated form in the plasma membrane 总被引:5,自引:2,他引:3
下载免费PDF全文
![点击此处可从《The Journal of cell biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
GTP-binding proteins which participate in signal transduction share a common heterotrimeric structure of the alpha beta gamma-type. In the activated state, the alpha subunit dissociates from the beta gamma complex but remains anchored in the membrane. The alpha subunits of several GTP-binding proteins, such as Go and Gi, are myristoylated at the amino terminus (Buss, J. E., S. M. Mumby, P. J. Casey, A. G. Gilman, and B. M. Sefton. 1987. Proc. Natl. Acad. Sci. USA. 84:7493-7497). This hydrophobic modification is crucial for their membrane attachment. The absence of fatty acid on the alpha subunit of Gs (Gs alpha), the protein involved in adenylate cyclase activation, suggests a different mode of anchorage. To characterize the anchoring domain of Gs alpha, we used a reconstitution model in which posttranslational addition of in vitro-translated Gs alpha to cyc- membranes (obtained from a mutant of S49 cell line which does not express Gs alpha) restores the coupling between the beta-adrenergic receptor and adenylate cyclase. The consequence of deletions generated by proteolytic removal of amino acid sequences or introduced by genetic removal of coding sequences was determined by analyzing membrane association of the proteolyzed or mutated alpha chains. Proteolytic removal of a 9-kD amino-terminal domain or genetic deletion of 28 amino-terminal amino acids did not modify the anchorage of Gs alpha whereas proteolytic removal of a 1-kD carboxyterminal domain abolished membrane interaction. Thus, in contrast to the myristoylated alpha subunits which are tethered through their amino terminus, the carboxy-terminal residues of Gs alpha are required for association of this protein with the membrane. 相似文献
14.
Highly Sensitive Immunoassay for the α Subunit of the GTP-Binding Protein Go and Its Regional Distribution in Bovine Brain 总被引:3,自引:2,他引:1
Tomiko Asano Reiji Semba Nobuaki Ogasawara Kanefusa Kato 《Journal of neurochemistry》1987,48(5):1617-1623
Antisera were raised in rabbits against the alpha subunit of a GTP-binding protein, Go. Because the antisera cross-reacted weakly with the alpha subunit of inhibitory GTP-binding protein of adenylate cyclase (Gi), they were purified with a Go alpha-coupled Sepharose column. Purified antibodies reacted only with Go alpha and did not cross-react with the Gi alpha subunit or beta gamma subunits in an immunoblot assay. Using these purified antibodies, a highly sensitive enzyme immunoassay method for the quantification of bovine brain Go alpha was developed. The assay system consisted of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody Fab' fragments labeled with beta-D-galactosidase from Escherichia coli. The minimal detection limit of the assay was 0.1 fmol, or 4 pg. The assay was specific for Go alpha, and it did not cross-react with Gi alpha or beta gamma. Samples from various regions of bovine brain were solubilized with 2% sodium cholate and 1 M NaCl, and the concentrations of Go alpha were determined. Go alpha was detected in all the regions, and the highest concentration was observed in the cerebral cortex. The immunohistochemical study showed that the neuropil was rich in Go alpha. 相似文献
15.
Molecular cloning and DNA sequence analysis of the human guanine nucleotide-binding protein Go alpha 总被引:4,自引:0,他引:4
S Lavu J Clark R Swarup K Matsushima K Paturu J Moss H F Kung 《Biochemical and biophysical research communications》1988,150(2):811-815
The nucleotide sequence of human Go alpha was determined from a partial human brain cDNA clone and the sequence of the first two 5' coding exons of a human genomic Go alpha clone. Comparison of this sequence with bovine and rat Go alpha shows greater than 90% homology at the nucleotide and deduced amino acid level. There is 100% identity at the amino acid level for the cholera and pertussis toxin-catalyzed ADP ribosylation sites, the putative guanine nucleotide binding, and the GTP hydrolysis sites. 相似文献
16.
R Morishita T Asano K Kato H Itoh Y Kaziro 《Biochemical and biophysical research communications》1989,161(3):1280-1285
Two alpha subunits of GTP-binding proteins were purified from bovine spleen membranes. Both proteins were ADP-ribosylated by pertussis toxin in the presence of beta gamma subunits. The major protein had a molecular mass of 40 kDa and its immunological reactivity and fragmentation pattern by limited proteolysis were identical with those of the alpha subunit of Gi2. The minor protein had a molecular mass of 41 kDa and its partial amino acid sequences completely matched with those predicted from human and rat Gi3 alpha cDNAs. 相似文献
17.
Y Nobukuni H Mitsubuchi F Endo J Asaka R Oyama K Titani I Matsuda 《Biochemistry》1990,29(5):1154-1160
A 1.7-kb cDNA clone encoding the entire precursor of the E1 beta subunit of the branched-chain alpha-ketoacid dehydrogenase (BCKDH) complex was isolated from a bovine liver cDNA library by screening with a mixture of synthetic oligonucleotide probes corresponding to the C-terminal five-residue sequence of the mature E1 beta subunit. A partial amino acid sequence was determined by Edman degradation of the intact subunit and the peptides generated by cleavage at the lysyl bonds. Nucleotide sequence analysis revealed that the isolated cDNA clone contained the 5'-untranslated sequence of 186 nucleotides, the translated sequence of 1176 nucleotides, and the 3'-untranslated sequence of 306 nucleotides with a poly(A) tail. A type AATAAA polyadenylation signal was located 17 nucleotides upstream of the start of a poly(A) tail. Comparison of the amino acid sequence predicted from the nucleotide sequence of the cDNA insert of the clone with the partial amino acid sequence of the mature BCKDH E1 beta subunit showed that the cDNA insert encodes for a 342 amino acid subunit with Mr 37,745 and that the subunit is synthesized as the precursor with a leader sequence of 50 amino acids and processed at the N-terminus. Northern blot analysis using the cDNA insert as a probe showed the presence of a 1.8-1.9-kb mRNA in bovine liver, suggesting that the insert covers nearly a full length of mRNA. Alignment of the deduced amino acid sequence of bovine BCKDH E1 beta with that of the human pyruvate dehydrogenase (PDH) complex E1 beta subunit revealed a high degree of sequence homology throughout the two enzymes.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
18.
R Morishita Y Fukada K Kokame T Yoshizawa K Masuda M Niwa K Kato T Asano 《European journal of biochemistry》1992,210(3):1061-1069
Heterotrimeric guanine-nucleotide-binding regulatory proteins (G proteins) have been classified into several subtypes on the basis of the properties of their alpha subunits, though a notable multiplicity of gamma subunits has also been demonstrated. To investigate whether each subtype of alpha subunit is associated with a particular gamma subunit, various oligomeric G proteins, purified from bovine tissues, were subjected to gel electrophoresis in a Tricine buffer system. All G proteins examined were shown to have more than two kinds of gamma subunit. Of the brain G proteins, GoA, GoB, and Gi1 contain the same set of three gamma subunits, but Gi2 contains only two of these subunits. Lung Gi1 and Gi2 and spleen Gi2 and Gi3 had similar sets of two gamma subunits, one of which was distinct from the gamma subunits of brain G proteins. These observations indicate that each subtype of alpha subunit is associated with a variety of beta gamma subunits, and that the combinations differ among cells. For analyses of the structural diversity of the gamma subunits, beta gamma subunits were purified from the total G proteins of each tissue and subjected to reverse-phase HPLC under denaturing conditions, where none of the beta subunits were eluted from the column. Three distinct gamma subunits were isolated in this way from brain beta gamma subunits. In contrast, lung and spleen beta gamma subunits contained at least five gamma subunits, the elution positions and electrophoretic mobilities of which were indistinguishable between the two tissues. Among several gamma subunits, two subspecies appeared to be common to the three tissues. In fact, in each case, the partial amino acid sequence of the most abundant gamma subunit in each tissue was identical, and the sequences coincided exactly with that of 'gamma 6' [Robishaw, J. D., Kalman, V. K., Moomaw, C. R. & Slaughter, C. A. (1989) J. Biol. Chem. 264, 15758-15761]. Fast-atom-bombardment mass spectrometry analysis indicated that this abundant gamma subunit in lung and spleen was geranylgeranylated and carboxymethylated at the C-terminus, as was 'gamma 6' from brain. In addition to abundant gamma subunits, other tissue-specific gamma subunits were also shown to be geranylgeranylated by gas-chromatography-coupled mass spectrometry analysis of Raney nickel-treated gamma subunits. These results suggest that most gamma subunits associated with many different subtypes of alpha subunit are geranylgeranylated in a variety of tissues, with the single exception being the retina where the G protein transducin has a farnesylated gamma subunit. 相似文献
19.
Isoprenylation of C-terminal cysteine in a G-protein gamma subunit 总被引:11,自引:0,他引:11
The predicted amino acid sequences for the Gi alpha 1 and G gamma 6 subunits of brain heterotrimeric G-proteins both contain C-terminal Cys-A-A-X elements (A is an aliphatic residue and X is any amino acid). This domain represents the site of Cys thioether modification by isoprenoids in p21ras, nuclear lamins, and fungal mating factors. We now show that G gamma 6, translated in reticulocyte lysate, is efficiently labeled with the isoprenoid precursor, [3H]mevalonate. Alteration of the sequence of G gamma 6 so that a Gly was substituted for Cys in the C-terminal Cys-A-A-X element rendered the protein incapable of undergoing isoprenoid modification. In contrast to G gamma 6, the Gi alpha 1 subunit did not appear to undergo isoprenylation when translated in reticulocyte lysate. Transient expression of the protein in COS cells, which were able to isoprenylate the p21 product of transfected H-ras, also failed to demonstrate isoprenylation of Gi alpha 1. The modification of the gamma subunit by a hydrophobic moiety may have important implications for the assembly of the brain G-protein beta gamma complexes into the cell membrane. 相似文献
20.
Relationships within the family of GTP-binding proteins isolated from bovine central nervous system 总被引:16,自引:0,他引:16
Four members of a family of GTP-binding proteins (G-proteins) which translate stimulation of extracellular receptors into regulation of intracellular enzymes were isolated from the bovine central nervous system. These proteins were examined for functional similarities and cross-reactivity with antibodies to the G-protein (transducin, Gt) from the photoreceptor system. Two proteins, Gs and Gi, can be distinguished by their respective abilities to stimulate or inhibit adenylate cyclase. The activated alpha subunits of Gt and a fourth member of the family, Go, did not affect this enzyme. Gt was shown to be unique in its ability to stimulate cGMP-dependent phosphodiesterase. While functionally diverse, the G-proteins were shown to have some common antigenic properties. Antibodies directed against the beta subunit of Gt recognize the beta 36 subunits of all preparations but not a putative second beta 35 subunit. Antibodies specific for the alpha subunit of Gt did not recognize other alpha subunits when immune blots from sodium dodecyl sulfate gels were examined. However, Go alpha, but not Gs alpha or Gi alpha, reacted strongly with the antibodies when the native subunit was spotted directly. This suggests that Go alpha and Gt alpha have homologous structural determinants. An antiserum that recognized Gt gamma did not recognize gamma subunits from other sources. These data support the proposed diversity of function and similarity of structure among the four G-proteins. The alpha and potentially gamma subunits appear to be responsible for the specificity of function. 相似文献