scholarly journals Detection of 23-27 kDa GTP-binding proteins in platelets and other cells

1987 ◽  
Vol 245 (2) ◽  
pp. 617-620 ◽  
Author(s):  
R P Bhullar ◽  
R J Haslam
Keyword(s):  
Binding Proteins ◽  
Soluble Fraction ◽  
Polyacrylamide Gel Electrophoresis ◽  
Human Platelets ◽  
Soluble Form ◽  
Rat Tissues ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Membrane Bound ◽  
Red Cell Membranes

Membrane proteins from rabbit and human platelets were separated by SDS/polyacrylamide-gel electrophoresis and the resolved polypeptides blotted on nitrocellulose. A family of GTP-binding proteins, termed Gn proteins, was detected by incubation of these blots with [alpha-32P]GTP in the presence of Mg2+. A major Gn protein with a molecular mass of 27 kDa (Gn27) and lesser amounts of 23, 24 and 25 kDa Gn proteins were observed in platelet membranes; much smaller amounts were in the platelet soluble fraction. Binding of [alpha-32P]GTP by platelet Gn proteins was blocked by GDP, GTP or guanosine 5′-[gamma-thio]triphosphate, but not by GMP or adenosine 5′-[beta gamma-imido]triphosphate. Rabbit and human red-cell membranes contained only Gn27. When rat tissues were analysed for Gn proteins, the largest amounts were found in brain, which contained two membrane-bound forms (Gn27 and Gn26) and a soluble form (Gn26).

NOVEL GTP-BINDING PROTEINS OF CYTOSOLIC AND MEMBRANE FRACTIONS OF HUMAN PLATELETS

1987 ◽  
Author(s):  
Eduardo G Lapetina ◽  
Bryan R Reep ◽  
Luis Molina Y Vedia
Keyword(s):  
Phospholipase C ◽  
Binding Proteins ◽  
Binding Protein ◽  
Human Platelets ◽  
Trypsin Treatment ◽  
Gtp Binding Protein ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Sds Page ◽  
Membrane Bound

We have assessed the binding of (α-32P)GTP to platelet proteins from cytosolic and membrane fractions. Proteins were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose. Incubation of the nitrocellulose blots with (α-32p)GTP indicated the presence of specific and distinct GTP-binding proteins in cytosol and membranes. Binding was prevented by 10-100 nM GTP or GTPyS and by 100 nM GDP; binding was unaffected by 1 nM-1 μM ATP. One main GTP-binding protein (29.5 KDa) was detected in the membrane fraction while three others (29, 27, and 21 KDa) were detected in the soluble fraction. Two cytosolic GTP-binding proteins (29 and 27 KDa) were degraded by trypsin; another cytosolic protein (21 KDa) and the membrane-bound protein (29.5 KDa) were resistant to the action of trypsin. Treatment of intact platelets with trypsin or thrombin, followed by lysis and fractionation, did not affect the binding of (α-32P)GTP to the membrane-bound protein. GTPyS still stimulates phospholipase C in permeabilized platelets already preincubated with trypsin. This suggests that trypsin-resistant GTP-binding proteins might regulate phospholipase C stimulated by GTPyS. We have started to purify the membrane-bound, trypsin-resistant, GTP-binding protein. Purification includes 1 M NaCl extraction and the use of an FPLC system with successive phenyl superose and superose 12 columns.

scholarly journals Identification of the ral and rac1 Gene Products, Low Molecular Mass GTP-binding Proteins from Human Platelets

1989 ◽  
Vol 264 (28) ◽  
pp. 16383-16389
Author(s):  
P G Polakis ◽  
R F Weber ◽  
B Nevins ◽  
J R Didsbury ◽  
T Evans ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Binding Proteins ◽  
Human Platelets ◽  
Gene Products ◽  
Gtp Binding Proteins ◽  
Gtp Binding

Molecular cloning of the cDNA for stimulatory GDP/GTP exchange protein for smg p21s (ras p21-like small GTP-binding proteins) and characterization of stimulatory GDP/GTP exchange protein

1991 ◽  
Vol 11 (5) ◽  
pp. 2873-2880
Author(s):  
K Kaibuchi ◽  
T Mizuno ◽  
H Fujioka ◽  
T Yamamoto ◽  
K Kishi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Exchange Reaction ◽  
Binding Proteins ◽  
Bovine Brain ◽  
Rat Tissues ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Ras P21 ◽  
Exchange Protein

We have recently purified to near homogeneity the stimulatory GDP/GTP exchange protein for smg p21s (ras p21-like GTP-binding proteins) from bovine brain cytosol. This regulatory protein, named GDP dissociation stimulator (GDS), stimulates the GDP/GTP exchange reaction of smg p21s by stimulating the dissociation of GDP from and the subsequent binding of GTP to them. In this study, we have isolated and sequenced the cDNA of smg p21 GDS from a bovine brain cDNA library by using an oligonucleotide probe designed from the partial amino acid sequence of the purified smg p21 GDS. The cDNA has an open reading frame encoding a protein of 558 amino acids with a calculated Mr value of 61,066, similar to the Mr of 53,000 estimated for the purified smg p21 GDS by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sucrose density gradient ultracentrifugation. The isolated cDNA is expressed in Escherichia coli, and the encoded protein exhibits smg p21 GDS activity. smg p21 GDS is overall hydrophilic, but there are several short hydrophobic regions. The smg p21 GDS mRNA is present in bovine brain and various rat tissues. smg p21 GDS has low amino acid sequence homology with the yeast CDC25 and SCD25 proteins, which may regulate the GDP/GTP exchange reaction of the yeast RAS2 protein, but not with ras p21 GTPase-activating protein, the inhibitory GDP/GTP exchange proteins (GDP dissociation inhibitor) for smg p25A and rho p21s, and the beta gamma subunits of heterotrimeric GTP-binding proteins such as Gs and Gi.

scholarly journals Farnesylcysteine analogues inhibit store-regulated Ca2+ entry in human platelets: evidence for involvement of small GTP-binding proteins and actin cytoskeleton

2000 ◽  
Vol 347 (1) ◽  
pp. 183-192 ◽  
Author(s):  
Juan A. ROSADO ◽  
Stewart O. SAGE
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Polymerization ◽  
Binding Proteins ◽  
Selective Inhibition ◽  
Human Platelets ◽  
Cytochalasin D ◽  
Dependent Manner ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Prenylated Proteins

We have investigated the mechanism of Ca2+ entry into fura-2-loaded human platelets by preventing the prenylation of proteins such as small GTP-binding proteins. The farnesylcysteine analogues farnesylthioacetic acid (FTA) and N-acetyl-S-geranylgeranyl-L-cysteine (AGGC), which are inhibitors of the methylation of prenylated and geranylgeranylated proteins respectively, significantly decreased thrombin-evoked increases in intracellular free Ca2+ concentration ([Ca2+]i) in the presence, but not in the absence, of external Ca2+, suggesting a relatively selective inhibition of Ca2+ entry over internal release. Both these compounds and N-acetyl-S-farnesyl-L-cysteine, which had similar effects to those of FTA, also decreased Ca2+ entry evoked by the depletion of intracellular Ca2+ stores with thapsigargin. The inactive control N-acetyl-S-geranyl-L-cysteine was without effect. Patulin, an inhibitor of prenylation that is inert with respect to methyltransferases, also decreased store-regulated Ca2+ entry. Cytochalasin D, an inhibitor of actin polymerization, significantly decreased store-regulated Ca2+ entry in a time-dependent manner. Both cytochalasin D and the farnesylcysteine analogues FTA and AGGC inhibited actin polymerization; however, when evoking the same extent of decrease in actin filament formation, FTA and AGGC showed greater inhibitory effects on Ca2+ entry, indicating a cytoskeleton-independent component in the regulation of Ca2+ entry by small GTP-binding-protein. These findings suggest that prenylated proteins such as small GTP-binding proteins are involved in store-regulated Ca2+ entry through actin cytoskeleton-dependent and cytoskeleton-independent mechanisms in human platelets.

GTP-binding Proteins in Human Platelets

Platelets ◽  
1990 ◽  
Vol 1 (2) ◽  
pp. 67-79 ◽  
Author(s):  
K. Nagata ◽  
Y. Nozawa
Keyword(s):  
Binding Proteins ◽  
Human Platelets ◽  
Gtp Binding Proteins ◽  
Gtp Binding

scholarly journals Detection of GTP-binding proteins in purified derivatives of rough endoplasmic reticulum

1989 ◽  
Vol 262 (2) ◽  
pp. 497-503 ◽  
Author(s):  
J Lanoix ◽  
L Roy ◽  
J Paiement
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Rough Endoplasmic Reticulum ◽  
Binding Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cell Types ◽  
Electrophoretic Analysis ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Dog Pancreas

As a first step in determining the molecular mechanism of membrane fusion stimulated by GTP in rough endoplasmic reticulum (RER), we have looked for GTP-binding proteins. Rough microsomes from rat liver were treated for the release of ribosomes, and the membrane proteins were separated by SDS/polyacrylamide-gel electrophoresis. The polypeptides were then blotted on to nitrocellulose sheets and incubated with [alpha-32P]GTP [Bhullar & Haslam (1987) Biochem. J. 245, 617-620]. A doublet of polypeptides (23 and 24 kDa) was detected in the presence of 2 microM-MgCl2. Binding of [alpha-32P]GTP was blocked by 1-5 mM-EDTA, 10-10,000 nM-GTP or 10 microM-GDP. Either guanosine 5′-[gamma-thio]triphosphate or guanosine 5′-[beta gamma-imido]triphosphate at 100 nM completely inhibited binding, but ATP, CTP or UTP at 10 mciroM did not. Pretreatment of microsomes by mild trypsin treatment (0.5-10 micrograms of trypsin/ml, concentrations known not to affect microsomal permeability) led to inhibition of [alpha-32P]GTP binding, suggesting a cytosolic membrane orientation for the GTP-binding proteins. Two-dimensional gel-electrophoretic analysis revealed the 23 and 24 kDa [alpha-32P]GTP-binding proteins to have similar acid isoelectric points. [alpha-32P]GTP binding occurred to similar proteins of rough microsomes from rat liver, rat prostate and dog pancreas, as well as to a 23 kDa protein of rough microsomes from frog liver, but occurred to distinctly different proteins in a rat liver plasma-membrane-enriched fraction. Thus [alpha-32P]GTP binding has been demonstrated to two low-molecular-mass (approx. 21 kDa) proteins in the rough endoplasmic reticulum of several varied cell types.

scholarly journals Molecular cloning of the cDNA for stimulatory GDP/GTP exchange protein for smg p21s (ras p21-like small GTP-binding proteins) and characterization of stimulatory GDP/GTP exchange protein.

1991 ◽  
Vol 11 (5) ◽  
pp. 2873-2880 ◽  
Author(s):  
K Kaibuchi ◽  
T Mizuno ◽  
H Fujioka ◽  
T Yamamoto ◽  
K Kishi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Exchange Reaction ◽  
Binding Proteins ◽  
Bovine Brain ◽  
Rat Tissues ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Ras P21 ◽  
Exchange Protein

We have recently purified to near homogeneity the stimulatory GDP/GTP exchange protein for smg p21s (ras p21-like GTP-binding proteins) from bovine brain cytosol. This regulatory protein, named GDP dissociation stimulator (GDS), stimulates the GDP/GTP exchange reaction of smg p21s by stimulating the dissociation of GDP from and the subsequent binding of GTP to them. In this study, we have isolated and sequenced the cDNA of smg p21 GDS from a bovine brain cDNA library by using an oligonucleotide probe designed from the partial amino acid sequence of the purified smg p21 GDS. The cDNA has an open reading frame encoding a protein of 558 amino acids with a calculated Mr value of 61,066, similar to the Mr of 53,000 estimated for the purified smg p21 GDS by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sucrose density gradient ultracentrifugation. The isolated cDNA is expressed in Escherichia coli, and the encoded protein exhibits smg p21 GDS activity. smg p21 GDS is overall hydrophilic, but there are several short hydrophobic regions. The smg p21 GDS mRNA is present in bovine brain and various rat tissues. smg p21 GDS has low amino acid sequence homology with the yeast CDC25 and SCD25 proteins, which may regulate the GDP/GTP exchange reaction of the yeast RAS2 protein, but not with ras p21 GTPase-activating protein, the inhibitory GDP/GTP exchange proteins (GDP dissociation inhibitor) for smg p25A and rho p21s, and the beta gamma subunits of heterotrimeric GTP-binding proteins such as Gs and Gi.

scholarly journals Characterization of Membrane-Bound Small GTP-Binding Proteins from Nicotiana tabacum

1995 ◽  
Vol 108 (1) ◽  
pp. 59-67 ◽  
Author(s):  
T. Haizel ◽  
T. Merkle ◽  
F. Turck ◽  
F. Nagy
Keyword(s):  
Nicotiana Tabacum ◽  
Binding Proteins ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Membrane Bound
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