scholarly journals The adipocyte Goα-immunoreactive polypeptide is different from the α subunit of the brain Go protein

1989 ◽  
Vol 260 (1) ◽  
pp. 307-310 ◽  
Author(s):  
B Rouot ◽  
J Carrette ◽  
M Lafontan ◽  
P Lan Tran ◽  
J A Fehrentz ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Molecular Mass ◽  
G Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Mass Range ◽  
Alpha Subunit ◽  
Α Subunit ◽  
Positive Immunoreactivity ◽  
Rat Adipose Tissue ◽  
The Brain

Rat adipose tissue possesses two Bordetella pertussis toxin (PTX) substrates and, in the same 39-41 kDa molecular mass range, positive immunoreactivity has also been reported with antibodies against the alpha subunit of Go, the major brain GTP-binding protein (G-protein). In this study, the presence of the brain Go alpha subunit at 39 kDa in adipocytes was reassessed, since direct correspondence between PTX substrates and Go alpha immunoreactivity has not yet been clearly established. On resolutive SDS/polyacrylamide-gel electrophoresis, the PTX substrates of human adipocytes were compared with the three PTX substrates found in brain. No ADP-ribosylated substrate at the level of the 39 kDa brain Go alpha could be detected in adipocyte membranes. Immunoblotting of human adipocyte membranes stained with our anti-Go alpha antibodies confirmed the presence of a positive immunoreactivity in this tissue, but the apparent molecular mass of the immunoreactive polypeptide in adipocytes was higher than that found in nervous tissues. Taken together, these results indicate that the brain Go alpha subunit is not present in adipose tissue. They also suggest the existence of a G-protein in adipocytes which is immunologically related to Go alpha but having a slightly higher molecular mass.

scholarly journals Impaired noradrenaline-induced lipolysis in white fat of aP2-Ucp1 transgenic mice is associated with changes in G-protein levels

2002 ◽  
Vol 364 (2) ◽  
pp. 369-376 ◽  
Author(s):  
Pavel FLACHS ◽  
JiŘí NOVOTNÝ ◽  
Filip BAUMRUK ◽  
Kristina BARDOVÁ ◽  
Lenka BOUŘOVÁ ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Transgenic Mice ◽  
G Protein ◽  
Energy Status ◽  
Α Subunit ◽  
Fat Depots ◽  
Ucp1 Expression ◽  
White Fat ◽  
Stimulation Of

In vitro experiments suggest that stimulation of lipolysis by catecholamines in adipocytes depends on the energy status of these cells. We tested whether mitochondrial uncoupling proteins (UCPs) that control the efficiency of ATP production could affect lipolysis and noradrenaline signalling in white fat in vivo. The lipolytic effect of noradrenaline was lowered by ectopic UCP1 in white adipocytes of aP2-Ucp1 transgenic mice, overexpressing the UCP1 gene from the aP2 gene promoter, reflecting the magnitude of UCP1 expression, the impaired stimulation of cAMP levels by noradrenaline and the reduction of the ATP/ADP ratio in different fat depots. Thus only subcutaneous but not epididymal fat was affected. UCP1 also down-regulated the expression of hormone-sensitive lipase and lowered its activity, and altered the expression of trimeric G-proteins in adipocytes. The adipose tissue content of the stimulatory G-protein α subunit was increased while that of the inhibitory G-protein α subunits decreased in response to UCP1 expression. Our results support the idea that the energy status of cells, and the ATP/ADP ratio in particular, modulates the lipolytic effects of noradrenaline in adipose tissue in vivo. They also demonstrate changes at the G-protein level that tend to overcome the reduction of lipolysis when ATP level in adipocytes is low. Therefore, respiratory uncoupling may exert a broad effect on hormonal signalling in adipocytes.

scholarly journals Hormonal regulation of Gi2 α-subunit phosphorylation in intact hepatocytes

1990 ◽  
Vol 268 (2) ◽  
pp. 449-457 ◽  
Author(s):  
M Bushfield ◽  
G J Murphy ◽  
B E Lavan ◽  
P J Parker ◽  
V J Hruby ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Protein Kinase ◽  
G Protein ◽  
Hormonal Regulation ◽  
Dose Effect ◽  
Alpha Subunit ◽  
Intact Cells ◽  
Α Subunit ◽  
Guanine Nucleotide Binding ◽  
Dose Dependent

Hepatocytes contain the Gi2 and Gi3 forms of the ‘Gi-family’ of guanine-nucleotide-binding proteins (G-proteins), but not Gi1. The anti-peptide antisera AS7 and I3B were shown to immunoprecipitate Gi2 and Gi3 selectively, and the antiserum CS1 immunoprecipitated the stimulatory G-protein Gs. Treatment of intact, 32P-labelled hepatocytes with one of glucagon, TH-glucagon ([1-N-alpha-trinitrophenylhistidine, 12-homoarginine]glucagon), Arg-vasopressin, angiotensin-II, the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate) and 8-bromo-cyclic AMP elicited a time- and dose-dependent increase in the labelling of the alpha-subunit of immunoprecipitated Gi2 which paralleled the loss of ability of low concentrations of the non-hydrolysable GTP analogue guanosine 5′-[beta gamma-imido]triphosphate (p[NH]ppG) to inhibit forskolin-stimulated adenylate cyclase activity (‘Gi’-function). The immunoprecipitation of phosphorylated Gi-2 alpha-subunit by the antiserum AS7 was blocked in a dose-dependent fashion by the inclusion of the C-terminal decapeptide of transducin, but not that of Gz (a ‘Gi-like’ G-protein which lacks the C-terminal cysteine group which is ADP-ribosylated by pertussis toxin in other members of the Gi family), in the immunoprecipitation assay. No labelling of the alpha-subunits of either Gi3 or Gs was observed. alpha-Gi2 was labelled in the basal state and this did not change over 15 min in the absence of ligand addition. In contrast to the monophasic dose-effect curves seen with vasopressin, angiotensin and TPA, the dose-effect curve for the glucagon-mediated increase in the labelling of alpha-Gi2 was markedly biphasic where the loss of Gi function paralleled the high-affinity component of the labelling of alpha-Gi2 caused by glucagon. TPA, TH-glucagon, angiotensin-II and vasopressin achieved similar maximal increases in the labelling of alpha-Gi2, which was approximately half that found after treatment of hepatocytes with either high glucagon concentrations (1 microM) or 8-bromocyclic AMP. Analysis of the phosphoamino acid content of immunoprecipitated alpha-Gi2 showed the presence of phosphoserine only. Incubation of hepatocyte membranes with [gamma-32P]ATP and purified protein kinase C, but not protein kinase A, led to the incorporation of label into immunoprecipitated alpha-Gi2. This labelling was abolished if membranes were obtained from cells which had received prior treatment with ligands shown to cause the phosphorylation of alpha-Gi2 in intact cells. We suggest that there are two possible sites for the phosphorylation of alpha-Gi2; one for C-kinase and the other for an unidentified kinase whose action is triggered by A-kinase activation.

scholarly journals Detection of Schistosoma mansoni Membrane Antigens by Immunoblot Analysis of Sera of Patients from Low-Transmission Areas

2005 ◽  
Vol 12 (2) ◽  
pp. 280-286 ◽  
Author(s):  
Italo M. Cesari ◽  
Diana E. Ballen ◽  
Leydi Mendoza ◽  
César Matos
Keyword(s):  
Schistosoma Mansoni ◽  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Surface Membrane ◽  
Sodium Dodecyl ◽  
Mass Range ◽  
Confirmatory Test ◽  
Low Transmission ◽  
Membrane Antigens

ABSTRACT Schistosoma mansoni surface membrane components play a relevant role in the host-parasite interaction, and some are released in vivo as circulating antigens. n-Butanol extraction favors the release of membrane antigens like alkaline phosphatase, which has been shown to be specifically recognized by antibodies from S. mansoni-infected humans and animals. In the present study, components in the n-butanol extract (BE) of the adult S. mansoni worm membrane fraction were separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D SDS-PAGE [15%]) and further analyzed by immunoblotting (immunoglobulin G) using defined sera. S. mansoni-infected patient sera, but not sera of uninfected patients or sera obtained from patients infected with other parasite species, specifically and variably recognized up to 20 polypeptides in the molecular mass range of ∼8 to >80 kDa. There were some differences in the number, intensity, and frequency of recognition of the BE antigens among sera from Venezuelan sites of endemicity with a different status of schistosomiasis transmission. Antigens in the 28- to 24-kDa molecular mass range appeared as immunodominants and were recognized by S. mansoni-positive sera from all the sites, with recognition frequencies varying between 57.5 and 97.5%. Immunoblotting with BE membrane antigens resulted in a highly sensitive (98.1%), specific (96.1.0%), and confirmatory test for the immunodiagnosis of schistosomiasis in low-transmission areas.

scholarly journals Prolyl 4-hydroxylase from Volvox carteri. A low-Mr enzyme antigenically related to the α subunit of the vertebrate enzyme

1988 ◽  
Vol 256 (1) ◽  
pp. 257-263 ◽  
Author(s):  
D D Kaska ◽  
R Myllylä ◽  
V Günzler ◽  
A Gibor ◽  
K I Kivirikko
Keyword(s):  
Green Alga ◽  
Polyacrylamide Gel Electrophoresis ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Affinity Column ◽  
Major Protein ◽  
Volvox Carteri ◽  
Chlamydomonas Reinhardii ◽  
Α Subunit ◽  
Major Protein Band

Prolyl 4-hydroxylase was isolated in a highly purified form from a multi-cellular green alga, Volvox carteri, by a procedure consisting of ion-exchange chromatography and affinity chromatography on poly(L-hydroxyproline) coupled to Sepharose. Two other affinity-column procedures were also developed, one involving 3,4-dihydroxyphenylacetate and the other 3,4-dihydroxyphenylpropionate linked to Sepharose. The Km values of the Volvox enzyme for the co-substrates and the peptide substrate, as well as the inhibition constants for selected 2-oxoglutarate analogues, were similar to those of the enzyme from Chlamydomonas reinhardii, except that the Km for 2-oxoglutarate with the Volvox enzyme was 6-fold greater. The temperature optimum of the Volvox enzyme was also 10 degrees C higher. The apparent Mr of the Volvox enzyme by gel filtration was about 40,000, being similar to that reported for the Chlamydomonas enzyme but markedly lower than that of the vertebrate enzymes. A similar apparent Mr of about 40,000 was also found for prolyl 4-hydroxylase from the green alga Enteromorpha intestinalis, whereas the enzyme from various vascular plants gave an apparent Mr greater than 300,000. SDS/polyacrylamide-gel electrophoresis demonstrated in the highly purified Volvox enzyme the presence of a major protein band doublet with a Mr of about 65,000 and a minor doublet of Mr about 55,000-57,000. A polyclonal antiserum, prepared against the Mr-65,000 doublet, stained in immunoblotting the Mr-65,000 doublet as well as the alpha subunit, but not the beta subunit, of the vertebrate prolyl 4-hydroxylase. An antiserum against the beta subunit of the vertebrate enzyme stained in immunoblotting a Mr-50,000 polypeptide in a partially purified Volvox enzyme preparation, but did not stain either the Mr-65,000 or the Mr-55,000-57,000 doublet of the highly purified enzyme. The data thus suggest that the active Volvox carteri prolyl 4-hydroxylase is an enzyme monomer antigenically related to the alpha subunit of the vertebrate enzyme.

scholarly journals Human leucocyte glycosylasparaginase is an α/β-heterodimer of 19 kDa α-subunit and 17 and 18 kDa β-subunit

1994 ◽  
Vol 300 (2) ◽  
pp. 541-544 ◽  
Author(s):  
O K Tollersrud ◽  
T Heiskanen ◽  
L Peltonen
Keyword(s):  
Molecular Mass ◽  
Reversed Phase ◽  
Alpha Subunit ◽  
Cross Linking ◽  
Terminal Sequence ◽  
Α Subunit ◽  
Sds Page ◽  
Specific Antisera ◽  
Purification Scheme ◽  
Chemical Cross Linking

Human lysosomal glycosylasparaginase (AGA; EC 3.5.1.26) consists of two glycosylated subunits, alpha and beta. Treatment with 3% SDS at 45 degrees C as part of a new purification scheme did not affect enzyme activity, but the alpha-subunit migrated an apparent 19 kDa peptide on SDS/PAGE instead of as a 24 kDa peptide, as observed without this SDS treatment. The N-terminal sequence was similar to that of the 24 kDa form, and, after reversed-phase h.p.l.c., the 19 kDa form was transformed to an apparent 24 kDa peptide on SDS/PAGE, indicating that their primary structures were identical. As the molecular mass of the alpha-subunit deduced from its cDNA was 19.5 kDa, the variation might be due to incomplete SDS coating of the 24 kDa form. This was confirmed by the tendency of the 24 kDa variant to polymerize even in the presence of SDS. The molecular mass of the beta-subunit was 17 and 18 kDa in accordance with previous reports. Chemical cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide resulted in the appearance of a 38 kDa peptide on SDS/PAGE which reacted with both the subunit-specific antisera on Western-blot analysis. On SDS/PAGE at pH 10.2 the active enzyme migrated as an apparent 43 kDa peptide. These results indicate that native human glycosylasparaginase is a heterodimer.

Immunological demonstration of Gq-protein in Limulus photoreceptors

1997 ◽  
Vol 14 (2) ◽  
pp. 287-292 ◽  
Author(s):  
Marlies Dorlöchter ◽  
Monika Klemeit ◽  
Hennig Stieve
Keyword(s):  
Molecular Mass ◽  
G Protein ◽  
G Proteins ◽  
Frozen Sections ◽  
Western Blots ◽  
Α Subunit ◽  
Gold Particles ◽  
Lateral Eye ◽  
Ultrathin Sections ◽  
Phototransduction Cascade

AbstractThe phototransduction cascade in invertebrates involves the coupling of rhodopsin activation to the action of the enzyme phospholipase C. This step is performed by G-proteins. An antibody against the α-subunit of a mouse Gq type G-protein recognized protein bands in Western blots of lateral eye and ventral nerve photoreceptors of Limulus. The protein bands had an apparent molecular mass of about 42 kDa. The antibody also recognized protein bands of a similar molecular mass in immunoblots of brain and intestine tissue. Immunoreactivity was found in lateral eye frozen sections where it was confined to the rhabdom region. When the antibody was applied to ultrathin sections of ventral nerve photoreceptors, the highest density of labeling was found on the rhabdomeral microvilli, but gold particles were also scattered in the cytoplasm. We conclude that a G-protein of the type Gq participates in the phototransduction of Limulus.

scholarly journals Studies on the α-subunit of bovine brain S-100 protein

1984 ◽  
Vol 218 (3) ◽  
pp. 691-696 ◽  
Author(s):  
H R Masure ◽  
J F Head ◽  
H M Tice
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fluorescence Emission ◽  
Bovine Brain ◽  
Alpha Subunit ◽  
Tryptophan Residue ◽  
Affinity Column ◽  
Apparent Dissociation Constant ◽  
Α Subunit ◽  
S 100

A method is described for the rapid purification of both S-100 protein and calmodulin from crude bovine brain extracts by the use of a fluphenazine-Sepharose affinity column eluted stepwise with decreasing concentrations of free Ca2+. Protein containing only alpha-subunit was purified from preparations of S-100 protein by anion-exchange chromatography. This protein co-migrated with the alpha-subunit of S-100 protein on sodium dodecyl sulphate/urea/polyacrylamide-gel electrophoresis and had an amino acid composition identical with that previously reported for this subunit. The results of u.v.-absorption and fluorescence-emission spectroscopy indicate that the tryptophan residue of the purified alpha-subunit of S-100 protein undergoes a Ca2+-induced change in environment. Measurements of changes in tryptophan fluorescence with increasing Ca2+ concentrations suggest an apparent dissociation constant of the alpha-subunit for Ca2+ of 7 × 10(-5)M in the absence of K+. In the presence of 90mM-K+ this value is increased to 3.4 × 10(-4)M.

scholarly journals The molecular cloning of the squid (Loligo forbesi) visual Gq-α subunit and its expression in Saccharomyces cerevisiae

1993 ◽  
Vol 292 (2) ◽  
pp. 333-341 ◽  
Author(s):  
N J P Ryba ◽  
J B C Findlay ◽  
J D Reid
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Cloning ◽  
G Protein ◽  
Alpha Subunit ◽  
Yeast Saccharomyces Cerevisiae ◽  
Α Subunit ◽  
Mating Response ◽  
Protein Functions ◽  
Loligo Forbesi ◽  
Photoreceptor Membranes

The sequence of the alpha-subunit of the major G-protein from the squid (Loligo forbesi) retina was predicted from its cDNA to be a member of the Gq subclass. The abundance of the squid Gq-alpha in the squid photoreceptor membranes suggests that the protein functions in phototransduction; the sequence of this G-protein is consistent with it mediating the light-dependent activation of a phospholipase C. The squid G-alpha was expressed in the yeast Saccharomyces cerevisiae, where it was unable to replace the function of GPA1, the yeast G-alpha homologue that regulates the mating response, suggesting that Gq-alpha was unable to interact with the endogenous G-beta gamma (STE4-STE18).

A compendium of G-protein–coupled receptors and cyclic nucleotide regulation of adipose tissue metabolism and energy expenditure

2020 ◽  
Vol 134 (5) ◽  
pp. 473-512 ◽  
Author(s):  
Ryan P. Ceddia ◽  
Sheila Collins
Keyword(s):  
Adipose Tissue ◽  
Energy Expenditure ◽  
Signaling Pathways ◽  
G Protein ◽  
Uncoupling Protein ◽  
Beige Adipocytes ◽  
Nucleotide Regulation ◽  
Ucp1 Expression ◽  
Thermogenic Adipocytes ◽  
G Protein Coupled

Abstract With the ever-increasing burden of obesity and Type 2 diabetes, it is generally acknowledged that there remains a need for developing new therapeutics. One potential mechanism to combat obesity is to raise energy expenditure via increasing the amount of uncoupled respiration from the mitochondria-rich brown and beige adipocytes. With the recent appreciation of thermogenic adipocytes in humans, much effort is being made to elucidate the signaling pathways that regulate the browning of adipose tissue. In this review, we focus on the ligand–receptor signaling pathways that influence the cyclic nucleotides, cAMP and cGMP, in adipocytes. We chose to focus on G-protein–coupled receptor (GPCR), guanylyl cyclase and phosphodiesterase regulation of adipocytes because they are the targets of a large proportion of all currently available therapeutics. Furthermore, there is a large overlap in their signaling pathways, as signaling events that raise cAMP or cGMP generally increase adipocyte lipolysis and cause changes that are commonly referred to as browning: increasing mitochondrial biogenesis, uncoupling protein 1 (UCP1) expression and respiration.

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