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PubMed1997

Qsr1p, a 60S ribosomal subunit protein, is required for joining of 40S and 60S subunits.

QSR1 is a recently discovered, essential Saccharomyces cerevisiae gene, which encodes a 60S ribosomal subunit protein. Thirty-one unique temperature-sensitive alleles of QSR1 were generated by regional codon randomization within a conserved 20-amino-acid sequence of the QSR1-encoded protein. The temperature-sensitive mutants arrest as viable, large, unbudded cells 24 to 48 h after a shift to 37 degrees C. Polysome and ribosomal subunit analysis by velocity gradient centrifugation of lysates from temperature-sensitive qsr1 mutants and from cells in which Qsr1p was depleted by down regulation of an inducible promoter revealed the presence of half-mer polysomes and a large pool of free 60S subunits that lack Qsr1p. In vitro subunit-joining assays and analysis of a mutant conditional for the synthesis of Qsr1p demonstrate that 60S subunits devoid of Qsr1p are unable to join with 40S subunits whereas 60S subunits that contain either wild-type or mutant forms of the protein are capable of subunit joining. The defective 60S subunits result from a reduced association of mutant Qsr1p with 60S subunits. These results indicate that Qsr1p is required for ribosomal subunit joining.

Cell Survival↗
PubMed1991

Effect of pepstatin A on structure and polymerization of intermediate filament subunit proteins in vitro.

Pepstatin A, a pentapeptide aspartyl protease inhibitor, can interact with intermediate filament (IF) subunit proteins and induce their polymerization in the absence of salt into long filaments with a rough surface and a diameter of 15-17 nm. This polymerization appears to be driven primarily by non-ionic interactions between pepstatin A and polymerization-competent forms of IF proteins, resulting in a composite filament. Proteolytic fragments of vimentin, lacking portions of only the head domain or of both the head and tail domains, failed to copolymerize with pepstatin A into long filaments under these conditions. Rather, these peptides, as well as control proteins like bovine serum albumin, were found to decorate pepstatin A polymers (filaments, ribbons, and sheets) by sticking to their surfaces. In addition to the electron microscopy experiments, UV difference spectra, ultracentrifugation, and SDS-PAGE analysis of in vitro cleavage products of vimentin obtained with HIV-1 protease all provided independent evidence for a direct association of pepstatin A with IF subunit proteins, with subsequent alterations in the IF subunit protein conformation. These data show that non-ionic interactions can substitute for the effect of salt and effectively drive the higher-order polymerization of IF subunit proteins.

Amino Acid Sequence↗
PubMed1996

The effect of GABA stimulation on GABAA receptor subunit protein and mRNA expression in rat cultured cerebellar granule cells.

1. After 8 days in vitro, rat cerebellar granule cells were exposed to 1 mM gamma-aminobutyric acid (GABA) for periods of 1, 2, 4, 6, 8 and 10 days. The effect of the GABA exposure on GABAA receptor alpha 1, alpha 6 and beta 2,3 subunit protein expression and alpha 1 and alpha 6 subunit steady-state mRNA levels, was examined using Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. 2. GABA exposure for 2 days decreased alpha 1 (35 +/- 10%, mean +/- s.e.mean), beta 2,3 (21 +/- 9%) and alpha 6 (28 +/- 10%) subunit protein expression compared to control levels. The GABA-mediated reduction in alpha 1 subunit expression after 2 days treatment was abolished in the presence of the GABAA receptor antagonist, Ru 5135 (10 microM). 3. GABA exposure for 8 days increased alpha 1 (26 +/- 10%, mean +/- s.e.mean) and beta 2,3 (56 +/- 23%) subunit protein expression over control levels, whereas alpha 6 subunit protein expression remained below control levels (by 38 +/- 10%). However, after 10 days GABA exposure, alpha 6 subunit protein expression was also increased over control levels by 65 +/- 29% (mean +/- s.e.mean). 4. GABA exposure did not change the alpha 1 or alpha 6 subunit steady-state mRNA levels over and 8 day period, nor did it alter the expression of cyclophilin mRNA over 1-8 days. 5. These results suggest that chronic GABA exposure of rat cerebellar granule cells has a bi-phasic effect on GABAA receptor subunit expression that is independent of changes to mRNA levels. Therefore, the regulation of the GABAA receptor expression by chronic agonist treatment appears to involve post-transcriptional and/or post-translational processes.

Amino Acid Isomerases↗
PubMed1980

30-S ribosomal subunit proteins of an Escherichia coli mutant in which assembly of the small ribosomal subunit is temperature-sensitive.

Escherichia coli 219ts2, a temperature-sensitive streptomycin-independent revertant of the streptomycin-dependent strain E. coli 209 is defective in 30-S ribosomal subunit assembly at 42 degrees C. Total 30-S ribosomal subunit proteins of this strain contain two additional components one of which (alpha) migrates below and the other (beta) to the right of protein S7 in two-dimensional polyacrylamide gel analyses carried out according to Kaltschmidt and Wittmann. These two components are also resolved from normal 30-S subunit proteins by chromatography on phosphocellulose. Tryptic fingerprinting of proteins alpha and beta identifies alpha as protein S7B, the form of S7 found in B strains of E. coli and beta as a mutant form of protein S4 produced by deletion of about 20 amino acids from the COOH terminus of the wild-type protein.

Amino Acids↗
PubMed2003

Structure of Mth11/Mth Rpp29, an essential protein subunit of archaeal and eukaryotic RNase P.

We have determined the solution structure of Mth11 (Mth Rpp29), an essential subunit of the RNase P enzyme from the archaebacterium Methanothermobacter thermoautotrophicus (Mth). RNase P is a ubiquitous ribonucleoprotein enzyme primarily responsible for cleaving the 5' leader sequence during maturation of tRNAs in all three domains of life. In eubacteria, this enzyme is made up of two subunits: a large RNA ( approximately 120 kDa) responsible for mediating catalysis, and a small protein cofactor ( approximately 15 kDa) that modulates substrate recognition and is required for efficient in vivo catalysis. In contrast, multiple proteins are associated with eukaryotic and archaeal RNase P, and these proteins exhibit no recognizable homology to the conserved bacterial protein subunit. In reconstitution experiments with recombinantly expressed and purified protein subunits, we found that Mth Rpp29, a homolog of the Rpp29 protein subunit from eukaryotic RNase P, is an essential protein component of the archaeal holoenzyme. Consistent with its role in mediating protein-RNA interactions, we report that Mth Rpp29 is a member of the oligonucleotide/oligosaccharide binding fold family. In addition to a structured beta-barrel core, it possesses unstructured N- and C-terminal extensions bearing several highly conserved amino acid residues. To identify possible RNA contacts in the protein-RNA complex, we examined the interaction of the 11-kDa protein with the full 100-kDa Mth RNA subunit by using NMR chemical shift perturbation. Our findings represent a critical step toward a structural model of the RNase P holoenzyme from archaebacteria and higher organisms.

Amino Acid Sequence↗
PubMed2001

Low expression of Galpha protein subunits in human prostate cancer.

PURPOSE: Because of increasing interest in G protein regulation of cell growth, differentiation and oncogenesis, we studied the functionality and expression of different G protein subunits in human prostate adenocarcinoma. MATERIALS AND METHODS: Surgical prostate specimens from control patients with bladder cancer and patients with prostate cancer were used. The functionality of alphas and alphai G protein subunits was evaluated by studying somatostatin or guanyl-5'-yl-imidotriphosphate regulation of forskolin stimulated adenylyl cyclase activity. The expression of alphas, alphai and beta subunits was studied by reverse transcriptase-polymerase chain reaction and immunoblot analysis. RESULTS: Adenylyl cyclase sensitivity to somatostatin inhibition decreased in prostate cancer. Low guanyl-5'-yl-imidotriphosphate doses inhibited forskolin stimulated adenylyl cyclase, whereas the opposite was true at high concentrations, evidencing the functionality of alphai and alphas, respectively, in normal and cancer tissue samples. Reverse transcriptase-polymerase chain reaction revealed RNA encoding for alphas and alphai1,2,3 subclasses in normal and pathological conditions. However, immunoblot analysis showed that the level of beta subunits was maintained, whereas that of alphas and alphai subunits decreased 30% to 40% after neoplastic transformation. The levels of alphas and alphai1,2 subunits correlated inversely with serum prostate specific antigen in patients with prostate cancer. CONCLUSIONS: The functionality and expression of G protein subunits are selectively modified in human prostate adenocarcinoma. Low alphas and alphai levels in prostate cancer suggest an important regulatory role of G proteins for cell proliferation and neoplastic transformation in the human prostate and they may have prognostic value.

Adenocarcinoma↗
PubMed1994

Regional characterization of G-protein subunits in glomeruli, cortices and medullas of the rat kidney.

We examined the types of guanine nucleotide-binding regulatory (G) protein subunits in isolated glomeruli, cortices excluding glomeruli and medullas of rat kidneys using bacterial toxin-catalyzed adenosine 5'-diphosphate (ADP) ribosylation and specific immunoblots. ADP ribosylation catalyzed by cholera or pertussis toxin revealed the presence of stimulatory G (Gs) or inhibitory G (Gi) proteins in membranes of the 3 segments of the kidney. Immunoblots further demonstrated the existence of several G-protein subunits, two Gs-protein alpha-subunits (G alpha s: 45 and 52 kD), Gi-protein alpha 1, alpha 2 and alpha 3-subunits (G alpha i1, G alpha i2: 40-41 kD, G alpha i3: 40 kD), bacterial toxin-insensitive G-protein alpha q- and alpha 11-subunits (G alpha q/11: 42 kD) and G-protein beta-subunits (G beta: 35-36 kD), in membranes of the preparations. The predominant subspecies of G alpha s was a 52-kD protein in glomerular membranes and a 45-kD protein in membranes of cortices and medullas. All of the G-protein subunits examined, however, were not detected in cytosolic fractions of glomeruli, cortices and medullas. Thus, we conclude that detectable quantities of several G-protein subunits including the new G-protein subunit, G alpha q/11, are present in membranes of glomeruli, cortices not containing glomeruli and medullas from the rat kidney. Both the existence of G alpha i1 and/or G alpha i2 subunits in glomeruli and the presence of G alpha q/11 subunits in the 3 preparations are new evidence.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate Ribose↗
PubMed1994

Spatial and temporal expression of the Dictyostelium discoideum G alpha protein subunit G alpha 2: expression of a dominant negative protein inhibits proper prestalk to stalk differentiation.

Previous results have shown that the G alpha protein subunit G alpha 2 is required for aggregation in Dictyostelium discoideum and is essential for coupling cell-surface cAMP receptors to downstream effectors in vivo during this stage of development. G alpha 2 expresses at least four distinct transcripts that are differentially regulated during development; two of the transcripts are expressed exclusively in the multicellular stages and their expression is restricted to prestalk cells. We partially dissected the G alpha 2 promoter and identified a component that is expressed exclusively during the multicellular stages using luciferase gene fusions. When this promoter region is coupled to lacZ, beta-gal expression is restricted to the multicellular stages and localized in prestalk cells with a pattern similar to that of the ecmA prestalk-specific promoter. We show that expression in wild-type cells of the G alpha 2 mutant protein [G alpha 2(G206T)] during the early stages of development blocks aggregation and cAMP-mediated activation of adenylyl cyclase and guanylyl cyclase, suggesting it functions as a dominant negatively active G alpha subunit. When this mutant G alpha protein is expressed from the ecmA prestalk-specific promoter, abnormal stalk differentiation during culmination is observed. Expression of the mutant G alpha 2 from the SP60 prespore promoter or wild-type G alpha 2 from either the ecmA or the SP60 promoter results in no detectable phenotype. The results suggest that G alpha 2 plays an essential role during the culmination stage in prestalk cells and may mediate cAMP receptor activation of these processes during multicellular development.

Amino Acid Sequence↗
PubMed1993

Bilateral ureteral obstruction alters levels of the G-protein subunits G alpha s and G alpha q/11.

To evaluate the effects of bilateral ureteral obstruction (BUO) on the levels of G-protein subunits in glomeruli, we examined the types and amounts of G-protein subunits in glomerular membranes from sham-operated control (SOC) rats and rats with BUO of 24 hours duration utilizing bacterial toxin-catalyzed ADP-ribosylation and specific antibodies. ADP-ribosylation catalyzed by cholera or pertussis toxin demonstrated the presence of Gs and Gi proteins in glomerular membranes. Immunoblots further revealed the existence of two types of G alpha s (45 and 52 kDa), as well as G alpha i2 (40 kDa), G alpha i3 (41 kDa), G alpha q/11 (42 kDa) and G beta (35 to 36 kDa) in glomerular membranes. The predominant subspecies of G alpha s was the 52 kDa protein. Detectable amounts of G alpha o were not found in glomerular membranes. Moreover, G-protein subunits were not detected in cytosolic extracts of glomeruli. Both forms of G alpha s and G alpha q/11 were significantly reduced in glomerular membranes from rats with BUO when compared to SOC rats. No significant difference in total G alpha i, G alpha i2 and G alpha i3 and G beta content was observed between the two groups of rats. In vivo pretreatment of rats with simultaneous administration of the angiotensin-converting enzyme inhibitor, enalaprilat, and the thromboxane synthase inhibitor, OKY-046, maintained the amount of G alpha s and G alpha q/11 in rats with BUO at the levels seen in SOC rats. The two drugs did not affect the amounts of G-protein subunits in glomerular membranes of SOC rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate Ribose↗
PubMed2003

[Correlation between basic expression level of NMDA receptor NR1 subunit protein in hippocampus and learning ability in rats].

OBJECTIVE: To investigate the relationship between basic expression level of NMDA receptor NR1 subunit protein in hippocampus and learning ability of rats. METHODS: Using a novel-object recognition model and Morris water maze,the novel-object recognition ability and spatial memory of SD rats were ranked, and grouped as the high (top 20 %) and the low (bottom 20%), respectively. NR1 subunit protein levels in hippocampus were measured by quantitative immunoblotting with NR1 subunit specific antibody. RESULT: The level of NR1 subunit protein in hippocampus in the high novel-object recognition ability group was 60% (P<0.01), higher than that in the low one, and in the high spatial memory group it was 45.4 % (P<0.05), higher than that in the low one, respectively. CONCLUSION: The basic expression level of NR1 subunit protein in hippocampus is related to novel-object recognition ability and spatial memory of rats.

Animals↗
PubMed1996

Differential localization and development-dependent expression of G-protein subunits, Go alpha and G beta, in rabbit heart.

Among various G-protein subunits identified in atrial and ventricular membranes from newborn and adult rabbits, the most remarkable developmental and tissue-specific differences were observed in the amounts of the alpha-subunit of Go (Go alpha) and the beta-subunit (G beta). Go alpha was abundant in atrial membranes especially from newborn rabbits but was barely detectable in ventricles. In contrast, G beta was present in both atrial and ventricular membranes. In both tissues the level of G beta-2 was much higher than that of G beta-1. Starting just after birth, the high levels of Go alpha in atria at term decreased gradually to about half of the amount in term animals, at 30 days of age, while the amount of G beta remained relatively constant until 26 days after birth. The levels of G beta in ventricles slowly declined during neonatal development. Similar developmental patterns were also observed when these parameters were studied in membranes isolated from cultured myocytes. Northern blot analysis showed the presence of mRNA for Go alpha-1A isoform in RNA samples from atria but not those from ventricles. The developmental expression of Go alpha-1A mRNA was somewhat different from that of Go alpha protein, whereas the time course of G beta mRNA expression was similar to that of G beta protein. Among other G-protein subunits tested, the amount of the splice variant of Gs protein, Gs alpha-1, in atrial as well as ventricular membranes increased with age of animals, while the amounts of Gs alpha-2, Gi alpha 2 and G alpha q/11 did not change significantly during development. These results indicate differential localization and tissue-specific developmental expression of G-protein subunits in rabbit heart.

Animals↗
PubMed2000

Dual regulation of Akt/protein kinase B by heterotrimeric G protein subunits.

While positive regulation of c-Akt (also known as protein kinase B) by receptor tyrosine kinases is well documented, compounds acting through G protein-coupled receptors can also activate Akt and its downstream targets. We therefore explored the role of G protein subunits in the regulation of Akt in cultured mammalian cells. In HEK-293 and COS-7 cells transiently transfected with beta(2)-adrenergic or m2 muscarinic receptors, respectively, treatment with agonist-induced phosphorylation of Akt at serine 473 as evidenced by phosphoserine-specific immunoblots. This effect was blocked by the phosphatidylinositol-3-OH kinase inhibitor LY294002 and wild-type Galpha(i1), and was not duplicated by co-transfection of the constitutively active Galpha(s)-Q227L or Galpha(i)-Q204L mutant. Co-transfection of Gbeta(1), Gbeta(2) but not Gbeta(5) together with Ggamma(2) activated the kinase when assayed in vitro following immunoprecipitation of the epitope-tagged enzyme. In contrast, constitutively activated G protein subunits representing the four Galpha subfamilies were found unable to activate Akt in either cell line. The latter results are in disagreement with a report by Murga et al. (Murga, C., Laguinge, L., Wetzker, R., Cuadrado, A., and Gutkind, J. S. (1998) J. Biol. Chem. 273, 19080-19085) that described activation of Akt in response to mutationally activated Galpha(q) and Galpha(i) transfection in COS cells. To the contrary, in our experiments Galpha(q)-Q209L inhibited Akt activation resulting from betagamma or mutationally activated H-Ras co-transfection in these cells. In HEK-293 cells Galpha(q)-Q209L transfection inhibited insulin-like growth factor-1 activation of epitope-tagged Akt. In m1 muscarinic receptor transfected HEK-293 cells, carbachol inhibited insulin-like growth factor-1 stimulated phosphorylation at Ser(473) of endogenous Akt in an atropine-reversible fashion. We conclude that G proteins can regulate Akt by two distinct and potentially opposing mechanisms: activation by Gbetagamma heterodimers in a phosphatidylinositol-3-OH kinase-dependent fashion, and inhibition mediated by Galpha(q). This work identifies Akt as a novel point of convergence between disparate signaling pathways.

Animals↗
PubMed1989

Electrophoretic and immunological comparisons of chloroplast and prokaryotic ribosomal proteins reveal that certain families of large subunit proteins are evolutionarily conserved.

Antibodies to individual chloroplast ribosomal (r-)proteins of Chlamydomonas reinhardtii synthesized in either the chloroplast or the cytoplasm were used to examine the relatedness of Chlamydomonas r-proteins to r-proteins from the spinach (Spinacia oleracea) chloroplast, Escherichia coli, and the cyanobacterium Anabaena 7120. In addition, 35S-labeled chloroplast r-proteins from large and small subunits of C. reinhardtii were co-electrophoresed on 2-D gels with unlabeled r-proteins from similar subunits of spinach chloroplasts, E. coli, and Anabaena to compare their size and net charge. Comigrating protein pairs were not always immunologically related, whereas immunologically related r-protein pairs often did not comigrate but differed only slightly in charge and molecular weight. In contrast, when 35S-labeled chloroplast r-proteins from large and small subunits of a closely related species C. smithii were coelectrophoresed with unlabeled C. reinhardtii chloroplast r-proteins, only one pair of proteins from each subunit showed a net displacement in mobility. Analysis of immunoblots of one-dimensional SDS and two-dimensional urea/SDS gels of large and small subunit r-proteins from these species revealed more antigenic conservation among the four species of large subunit r-proteins than small subunit r-proteins. Anabaena r-proteins showed the greatest immunological similarity to C. reinhardtii chloroplast r-proteins. In general, antisera made against chloroplast-synthesized r-proteins in C. reinhardtii showed much higher levels of cross-reactivity with r-proteins from Anabaena, spinach, and E. coli than did antisera to cytoplasmically synthesized r-proteins. All spinach r-proteins that cross-reacted with antisera to chloroplast-synthesized r-proteins of C. reinhardtii are known to be made in the chloroplast (Dorne et al. 1984b). Four E. coli r-proteins encoded by the S10 operon (L2, S3, L16, and L23) were found to be conserved immunologically among the four species. Two of the large subunit r-proteins, L2 and L16, are essential for peptidyltransferase activity. The third (L23) and two other E. coli large subunit r-proteins (L5 and L27) that have immunological equivalents among the four species are functionally related to but not essential for peptidyltransferase activity.

Blotting, Western↗
PubMed1996

G protein subunits and the stimulation of phospholipase C by Gs-and Gi-coupled receptors: Lack of receptor selectivity of Galpha(16) and evidence for a synergic interaction between Gbeta gamma and the alpha subunit of a receptor activated G protein.

Stimulatory guanine nucleotide binding protein (Gs)-coupled receptors activated by luteinizing hormone, vasopressin, and the catecholamine isoproterenol (luteinizing hormone receptor, type 2 vasopressin receptor, and types 1 and 2 beta-adrenergic receptors) and the Gi-coupled M2 muscarinic receptor (M2R) were expressed transiently in COS cells, alone and in combination with Gbeta gamma dimers, their corresponding Galphas (Galpha(s), or Galpha(i3)) and either Galpha(q) or Galpha(16). Phospholipase C (PLC) activity, assessed by inositol phosphate production from preincorporated myo[3H]inositol, was then determined to gain insight into differential coupling preferences among receptors and G proteins. The following were observed: (i) All receptors tested were able to stimulate PLC activity in response to agonist occupation. The effect of the M2R was pertussis toxin sensitive. (ii) While, as expected, expression of Galpha(q) facilitated an agonist-induced activation of PLC that varied widely from receptor to receptor (400% with type 2 vasopressin receptor and only 30% with M2R), expression of Galpha(16) facilitated about equally well the activation of PLC by any of the tested receptors and thus showed little if any discrimination for one receptor over another. (iii) Gbeta gamma elevated basal (agonist independent) PLC activity between 2- and 4-fold, confirming the proven ability of Gbeta gamma to stimulate PLCbeta. (iv) Activation of expressed receptors by their respective ligands in cells coexpressing excess Gbeta gamma elicited agonist stimulated PLC activities, which, in the case of the M2R, was not blocked by pertussis toxin (PTX), suggesting mediation by a PTX-insensitive PLC-stimulating Galpha subunit, presumably, but not necessarily, of the Gq family. (v) The effects of Gbeta gamma and the PTX-insensitive Galpha elicited by M2R were synergistic, suggesting the possibility that one or more forms of PLC are under conditional or dual regulation of G protein subunits such that stimulation by one sensitizes to the stimulation by the other.

Animals↗
PubMed2000

Structural differences between mesophilic, moderately thermophilic and extremely thermophilic protein subunits: results of a comprehensive survey.

BACKGROUND: Proteins from thermophilic organisms usually show high intrinsic thermal stability but have structures that are very similar to their mesophilic homologues. From prevous studies it is difficult to draw general conclusions about the structural features underlying the increased thermal stability of thermophilic proteins. RESULTS: In order to reveal the general evolutionary strategy for changing the heat stability of proteins, a non-redundant data set was compiled comprising all high-quality structures of thermophilic proteins and their mesophilic homologues from the Protein Data Bank. The selection (quality) criteria were met by 64 mesophilic and 29 thermophilic protein subunits, representing 25 protein families. From the atomic coordinates, 13 structural parameters were calculated, compared and evaluated using statistical methods. This study is distinguished from earlier ones by the strict quality control of the structures used and the size of the data set. CONCLUSIONS: Different protein families adapt to higher temperatures by different sets of structural devices. Regarding the structural parameters, the only generally observed rule is an increase in the number of ion pairs with increasing growth temperature. Other parameters show just a trend, whereas the number of hydrogen bonds and the polarity of buried surfaces exhibit no clear-cut tendency to change with growth temperature. Proteins from extreme thermophiles are stabilized in different ways to moderately thermophilic ones. The preferences of these two groups are different with regards to the number of ion pairs, the number of cavities, the polarity of exposed surface and the secondary structural composition.

Amino Acids↗
PubMed1993

Resonance energy transfer between guanine nucleotide binding protein subunits and membrane lipids.

Resonance energy transfer was used to estimate the distances of closest approach between fluorescent labels on G protein subunits (alpha, beta, and gamma) and the phospholipid bilayer surface. Fluorescein-labeled alpha, beta, and gamma subunits were the energy transfer donors and hexadecylaminoeosin (HAE) in phospholipid vesicles was the acceptor. Bovine brain G protein (alpha o, beta, and gamma subunits) were individually labeled with fluorescein as described in the accompanying paper [Kwon et al. (1993) Biochemistry (preceding paper in this issue)]. Fluorescein-labeled subunits were combined with the appropriate unlabeled G protein subunits and reconstituted into phospholipid vesicles. HAE quenched the fluorescein emission in a concentration-dependent manner which was dependent upon the G protein subunit labeled (gamma = beta > alpha o). From steady-state quenching data with hexadecylaminofluorescein (HAF) as a standard to determine the density of HAE in the phospholipid bilayer, the calculated distances between fluorescein-alpha o, -beta, and -gamma and HAE are 46 +/- 2, 38 +/- 3, and 37 +/- 2 A, respectively. Energy transfer was identified as the means of fluorescence quenching by two methods: (1) addition of 0.1% Lubrol reversed the quenching of fluorescein-labeled G protein subunits by HAE and (2) sensitized HAE emission was observed in the presence of fluorescein-labeled G protein subunits. These results are the first physical measurements of the distances between sites on G protein subunits and the lipid bilayer. These data demonstrate that the label in the beta and gamma subunits is closer to the lipid bilayer than that in the alpha subunit.

Animals↗
PubMed1995

Disproportionate regulation of nuclear- and mitochondrial-encoded cytochrome oxidase subunit proteins by functional activity in neurons.

Cytochrome oxidase is the terminal enzyme in the mitochondrial respiratory chain engaged in oxidative metabolism and energy production. In mammals, the holoenzyme is composed of 13 subunits encoded by both nuclear and mitochondrial genomes. The goal of the present study was to compare the effect of afferent impulse blockade on the expression of these two genomes at the subunit protein level. It also aimed to determine the correlation between the level of cytochrome oxidase activity and the relative amount of subunit proteins. Relative enzyme activity was analysed histochemically, and relative amounts of subunits IV (nuclear-encoded) and II/III (mitochondrial-derived) proteins were obtained immunohistochemically by anti-subunit IV and anti-subunit II/III antibodies in the lateral geniculate nucleus and the primary visual cortex of adult monkeys. In the normal visual centers, similar staining patterns were found for all three markers. After three and seven days of tetrodotoxin treatment, levels of enzyme activity and subunit proteins declined disproportionately in the deprived laminae of the visual center. Densitometric analysis indicates that changes in enzyme activity and subunit IV proteins were significantly greater than those of subunit II/III proteins (P < 0.01). The finding that nuclear and mitochondrial genomes are disproportionately regulated at subunit protein levels by neuronal activity implies that the two genomes operate under different regulatory mechanisms. Changes in subunit IV paralleled most closely those of cytochrome oxidase activity (coefficient of determination r2 = 0.95). This suggests that nuclear-derived subunit IV protein may play a pivotal role in controlling cytochrome oxidase holoenzyme activity.

Animals↗
PubMedDate not supplied

Analytical pharmacology of G protein-coupled receptors by stoichiometric expression of the receptor and G(alpha) protein subunits.

The description of a new family of recombinant proteins, which are constructed by the covalent fusion of the cDNA encoding a G protein-coupled receptor with that of a G(alpha) protein subunit, has recently been introduced as an original strategy to explore receptor pharmacology under defined experimental conditions. As such, a controlled 1:1 stoichiometry of receptor and G(alpha) protein expression can be achieved, as well as a forced spatial proximity to each other. Fusion proteins have been revealed as active at the receptor ligand binding level and functional at the G(alpha) protein and effector level. Insights on analytical pharmacological data are discussed for wild-type and mutant receptors interacting with a given G(alpha) protein subunit and different subtypes of either wild-type or mutant G(alpha) proteins activated by a single receptor subtype. A possible alteration of the receptor:G(alpha) protein selectivity may occur due either to the spatial proximity of both protein partners or to a constraint receptor state unable to accommodate to different G(alpha) protein states. Coactivation of endogenous G(alpha) proteins in host cells expressing a fusion protein has also been observed, but depends mainly on the coupling efficiency of the receptor and G(alpha) protein engaged in the fusion process. The ligand's apparent intrinsic activity has been shown to be either enhanced, attenuated, or unmodified when the functional responses of a fusion protein are compared to the coexpression of both fusion protein partners.

Animals↗