Disease relevance of GTP-Gamma-32P

• Experiments with pertussis and cholera toxins revealed that pretreating membranes with pertussis toxin (100 ng/ml) inhibited by 50% the IL1-induced [35S]GTP gamma S binding and [gamma-32P]GTP hydrolysis [1].
• Incubation of [alpha-32P]GTP with cellular extracts or membranes of Streptomyces coelicolor labels a protein of 43 kDa, which was also labeled with [8,5'-3H]GTP but not with [alpha-32P]ATP or [gamma-32P]GTP [2].
• ATP citrate lyase (EC 4.3.1.8.) was shown to be a major phosphorylation protein of a fraction derived from Zajdela rat hepatoma by chromatography on heparin-Ultrogel, after the incubation with [gamma-32P]ATP or [gamma-32P]GTP [3].
• Plasma-membrane-bound kinases of AS-30D ascites from transplantable rat hepatocarcinoma were shown to extensively catalyze the phosphorylation of plasma membrane proteins and membrane lipids, using [gamma-32P]ATP or [gamma-32P]GTP as a phosphate donor [4].

High impact information on GTP-Gamma-32P

• Constitutively activated RhoA, loaded with [gamma-32P]GTP, directly interacted with GST-IL-1Rcd in a filter-binding assay [5].
• By preloading recombinant Ran/TC4 with [gamma-32P]GTP or [3H]GDP, we show that the interactions with p97 and NTF2 are specific for the GTP- and GDP-bound forms, respectively [6].
• Lysate from quiescent platelets promotes rapid hydrolysis of [gamma-32P]GTP bound to rap1B [7].
• The S6 kinase in crude extracts utilized both [gamma-32P]ATP (apparent Km congruent to 6-8 microM) and [gamma-32P]GTP (apparent Km congruent to 3 microM), but the ability to utilize GTP was lost after partial purification of the kinase [8].
• In vitro, the valine form of p21 had 2.4- and 2.7-fold greater autophosphorylating activity than the glycine and arginine forms of p21, respectively, using [gamma-32P]GTP as phosphate donor, but the three p21 species had similar Km values for GTP (0.20-0.27 microM) [9].

Biological context of GTP-Gamma-32P

• Phosphorylation of native succinic thiokinase with [gamma-32P]ATP and [gamma-32P]GTP showed radioactivity only in the alpha monomer [10].
• Using additional information from the kinetics of 32Pi production and release from EF-Tu X Mn X [gamma-32P]GTP these were identified as EF-Tu X Mn X GTP (linewidth 4.2 mT), EF-Tu X Mn X GDP X Pi (1.20 mT) and EF-Tu X Mn X GDP (1.29 mT) [11].
• In addition, hybridization of RNA transcripts labelled at their 5' end by either [gamma32P]ATP or [gamma-32P]GTP indicated that not only elongation but also initiation occurred randomly through the entire adenovirus-2 genome, irrespective of the form of the enzyme and of the origin of the cells (normal or infected) [12].

Anatomical context of GTP-Gamma-32P

• One major substrate protein was phosphorylated with [gamma-32P]GTP in membranes of human leukemia (HL-60) cells [13].
• It nonetheless stereospecifically induced these responses in cells primed with tumor necrosis factor-alpha and likewise induced PMN plasma membranes to bind 35S-labeled guanosine 5'-O-(thiotriphosphate) (GTP gamma S) and phosphohydrolyze [gamma-32P]GTP [14].
• Phosphorylation of EIF2 by protein kinases present in reticulocyte lysates was demonstrated using [gamma-32P]GTP or [gamma-32P]ATP as the phosphate donor [15].
• To isolate target proteins involved in these signaling pathways, we have probed proteins from neutrophil cytosol immobilized on nitrocellulose with Cdc42Hs labeled with [gamma-32P]GTP [16].
• Highly purified endoplasmic reticulum fractions from rat liver and dog pancreas harbor membrane-associated kinases that phosphorylate integral membrane proteins of 90, 56, 35, and 15 kDa with [gamma-32P]GTP and of 90, 56, and 35 kDa with [gamma-32P]ATP [17].

Associations of GTP-Gamma-32P with other chemical compounds

• In purified sarcolemmal membranes, carbachol and a variety of other muscarinic receptor (MR) agonists induced increases in [3H]GTP, [gamma-32P]GTP, and [3H]GDP binding to relatively high affinity sites [18].
• The rate of the fluorescence decay matches the rate of [32P]Pi production due to [gamma-32P]GTP hydrolysis, and the decay is immediately reversed by rechallenging with GTP [19].
• The endogenous kinase was identified as casein kinase II (CK II) based on the inhibition of the endogenous phosphorylation 160/150-kDa proteins by heparin, 5.6-dichlorobenzimidazole riboside, polyaspartyl peptide and hemin, and its ability to use [gamma-32P]GTP as the phosphate donor [20].
• The enzyme incubated with [gamma 32P]ATP or [gamma 32P]GTP as phosphoryl donors undergoes autophosphorylation in the M(r) = 25,000 subunit [21].

Gene context of GTP-Gamma-32P

• To identify target proteins involved in these signaling pathways cell extracts immobilized on nitrocellulose have been probed with [gamma-32P]GTP-labeled Rac1, RhoA, and Cdc42Hs [22].
• For this purpose, cytosolic extracts were incubated with recombinant human p21ras complexed to [gamma-32P]GTP and the time-dependent decrease in p21ras bound radioactivity was measured [23].
• The phosphorylation system of the membrane was able to utilize [gamma-32P]GTP in both the basal and EGF-stimulated reactions [24].
• The rate of release of Pi from [gamma-32P]GTP-bound ram p25 was calculated to be 0.011 min-1 [25].
• However, under conditions that promoted GTP-dependent desensitization, there was no apparent phosphorylation of LH/CG receptor (obtained via immunoprecipitation) by endogenous membrane-associated protein kinases using [gamma-32P]GTP or [gamma-32P]ATP as phosphate donor [26].

Analytical, diagnostic and therapeutic context of GTP-Gamma-32P

• Endogenous substrates of the kinase were demonstrated by incubating sperm cytosolic extracts with [gamma-32P]GTP, under conditions that limit the expression of other protein kinases, and analyzing the products by SDS-PAGE and autoradiography [27].
• Using highly purified enzyme and [gamma-32P]GTP as the phosphate donor, the neutralized extract then serves as substrate for adenosine kinase, and the AMP product is purified by high-performance liquid chromatography [28].

References