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Query: UNIPROT:P06889 (Mol)
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We have previously shown that the polyethylene glycol conjugated superoxide dismutase (SOD), which has a plasma half-life of more than 24 h, protects the blood perfused rabbit heart against injury during ischaemia and reperfusion. However, the profile for the dose-dependency of protection was bell-shaped with loss of efficacy below 6000 and above 30,000 U/kg. In the present study, isolated rabbit hearts, perfused with blood from support rabbits, were subjected to a 2 min infusion with St Thomas' Hospital cardioplegic solution followed by 60 min of global ischaemia (37 degrees C) and 60 min of reperfusion. PEG-SOD was administered 1 h or 12-24 h before ischaemia. We assessed the effect of PEG-SOD on ischaemia- and reperfusion-induced changes in: (i) the tissue content of reduced glutathione (GSH), oxidized glutathione (GSSG) and malondialdehyde (MDA) and (ii) the activity of CuZn-SOD, Mn-SOD and glutathione peroxidase and reductase (GPD and GRD). Ischaemia and reperfusion reduced tissue GSH content by 70% and increased GSSG content by 400% (from their fresh aerobic values of 13.1.9 and 0.09 +/- 0.01 nmol/mg protein, respectively). PEG-SOD, given intravenously at various doses to donor and support rabbits 1 h or 12-24 h before ischaemia, protected against these changes with a bell-shaped dose-response relationship. Thus, with 0, 3000, 6000, 12,000, 30,000 and 60,000 U/kg, GSH content was 4.1 +/- 0.4, 4.8 +/- 0.4, 8.5 +/- 0.5, 12.3 +/- 1.6, 12.3 +/- 1.6 and 5.0 +/- 0.5 nmol/mg protein in the 1 h pretreatment group and 4.1 +/- 0.4, 4.2 +/- 0.5, 10.4 +/- 1.5, 11.2 +/- 1.1, 11.4 +/- 0.7 and 4.7 +/- 0.6 nmol/mg protein in the 12-24 h pretreatment group (means +/- S.E.M.). For GSSG the corresponding values were 0.36 +/- 0.04, 0.34 +/- 0.03, 0.12 +/- 0.01, 0.12 +/- 0.01, 0.11 +/- 0.01 and 0.41 +/- 0.03 nmol/mg protein for the 1 h group and 0.36 +/- 0.04, 0.35 +/- 0.02, 0.15 +/- 0.01, 0.12 +/- 0.01, 0.11 +/- 0.01 and 0.34 +/- 0.02 nmol/mg protein for the 12-24 h group. Ischaemia and reperfusion had no effect on tissue MDA content or CuZn-SOD, GDP and GRD activity, and in general, PEG-SOD also lacked significant effect on any of these variables at any dose studied. However, Mn-SOD activity was severely reduced by ischaemia and reperfusion (from 42 +/- 7 U/mg protein in fresh aerobic controls to 6 +/- 1 U/mg protein at the end of reperfusion).(ABSTRACT TRUNCATED AT 400 WORDS)
J Mol Cell Cardiol 1992 Sep
PMID:PEG-SOD and myocardial antioxidant status during ischaemia and reperfusion: dose-response studies in the isolated blood perfused rabbit heart. 143 18

Epidermal growth factor (EGF) can stimulate inositol lipid hydrolysis in rat hepatocytes and can accelerate GTP/GDP exchange in hepatic membranes. Both of these responses can be abolished by pretreatment with pertussis toxin, suggesting that EGF may regulate phospholipase C (PLC) activity via a guanine nucleotide-binding regulatory protein (G protein) in liver cells. In contrast, in A431 human epidermoid carcinoma cells EGF can induce a rapid phosphorylation of PLC-gamma on tyrosine residues that increases the activity of immunoprecipitated PLC-gamma, suggesting that tyrosine phosphorylation of PLC-gamma may be the mechanism for EGF-stimulated inositol trisphosphate production in these cells. To determine the importance of the phosphorylation of PLC-gamma on tyrosine residues in a system where the EGF receptor apparently couples to a G protein, the effect of EGF on tyrosine phosphorylation of PLC-gamma was examined in rat hepatocytes. PLC-gamma was immunoprecipitated from cell lysates with a PLC-gamma antiserum and its tyrosine phosphorylation state was determined using both Western blot analysis with phosphotyrosine antibodies and direct measurement of phosphorylated amino acids. The results were compared with analogous experiments performed with A431 cells and another cultured cell line expressing high levels of human EGF receptors, Rat1hER fibroblasts. Although the amount of PLC-gamma in rat hepatocytes is similar to that in A431 cells and slightly higher than that in Rat1hER cells, EGF causes a barely detectable increase in the phosphorylation of PLC-gamma on tyrosine in hepatocytes, whereas it stimulates a significant degree of phosphorylation of PLC-gamma on tyrosine in Rat1hER or A431 cells. Pretreatment of hepatocytes with pertussis toxin abolishes the ability of EGF to activate PLC, as determined by an increase in intracellular Ca2+, but has no effect on the small amount of phosphate incorporated into tyrosine residues on the PLC-gamma protein, demonstrating that this low level of PLC-gamma phosphorylation does not correlate with changes in PLC activity. The data suggest that phosphorylation of PLC-gamma on tyrosine is not important for EGF-enhanced PLC activity in hepatocytes. This conclusion implies that EGF may use a mechanism to regulate PLC activity in hepatocytes that is different from that used in cultured cells expressing high levels of EGF receptors.
Mol Pharmacol 1992 Nov
PMID:Epidermal growth factor activates phospholipase C in rat hepatocytes via a different mechanism from that in A431 or rat1hER cells. 143 49

The rap1A gene encodes a 21-kDa, ras-related GTP-binding protein (p21rap1A) of unknown function. A close structural homolog of p21rap1A (65% identity in the amino-terminal two-thirds) is the RSR1 gene product (Rsr1p) of Saccharomyces cerevisiae. Although Rsr1p is not essential for growth, its presence is required for nonrandom selection of bud sites. To assess the similarity of these proteins at the functional level, wild-type and mutant forms of p21rap1A were tested for complementation of activities known to be fulfilled by Rsr1p. Expression of p21rap1A, like multicopy expression of RSR1, suppressed the conditional lethality of a temperature-sensitive cdc24 mutation. Point mutations predicted to affect the localization of p21rap1A or its ability to cycle between GDP and GTP-bound states disrupted suppression of cdc24ts, while other mutations in the 61-65 loop region improved suppression. Expression of p21rap1A could not, however, suppress the random budding phenotype of rsr1 cells. p21rap1A also apparently interfered with the normal activity of Rsrlp, causing random budding in diploid wild-type cells, suggesting an inability of p21rap1A to interact appropriately with Rsr1p regulatory proteins. Consistent with this hypothesis, we found an Rsr1p-specific GTPase-activating protein (GAP) activity in yeast membranes which was not active toward p21rap1A, indicating that p21rap1A may be predominantly GTP bound in yeast cells. Coexpression of human Rap1-specific GAP suppressed the random budding due to expression of p21rap1A or its derivatives, including Rap1AVal-12. Although Rap1-specific GAP stimulated the GTPase of Rsr1p in vitro, it did not dominantly interfere with Rsr1p function in vivo. A chimera consisting of Rap1A1-165::Rsr1p166-272 did not exhibit normal Rsr1p function in the budding pathway. These results indicated that p21rap1A and Rsr1p share at least partial functional homology, which may have implications for p21rap1A function in mammalian cells.
Mol Cell Biol 1992 Sep
PMID:Functional interaction between p21rap1A and components of the budding pathway in Saccharomyces cerevisiae. 150 5

The biochemical properties of Artemia ras proteins (p21) have been studied after immunoprecipitation with the monoclonal antibody Y13-259. The ras products bind GTP and GDP, and have GTPase activity. Artemia p21 was unable to hydrolyze Gp4G, although this dinucleotide exhibits high affinity for the protein. Our results demonstrate that the protein(s) recognized by the Y13-259 antibody in this crustacean behave as typical mammalian ras p21s.
Mol Cell Biochem 1992 May 13
PMID:Biochemical characterization of Artemia ras p21. 151 32

Tubulin binds guanine nucleotides with high affinity and specificity. GTP, an allosteric effector of microtubule assembly, requires Mg2+ for its interaction with beta-tubulin and binds as the MgGTP complex. In contrast, GDP binding does not require Mg2+. The structural basis for this difference is not understood but may be of fundamental importance for microtubule assembly. We investigated the interaction of beta-tubulin with guanine nucleotides using site-directed mutagenesis. Acidic amino acid residues have been shown to interact with nucleotide in numerous nucleotide-binding proteins. In this study, we mutated seven highly conserved aspartic acid residues and one highly conserved glutamic acid residue in the putative GTP-binding domain of beta-tubulin (N-terminal 300 amino acids) to asparagine and glutamine, respectively. The mutants were synthesized in vitro using rabbit reticulocyte lysates, and their affinities for nucleotide determined by an h.p.l.c.-based assay. Our results indicate that the mutations can be placed in six separate categories on the basis of their effects on nucleotide binding. These categories range from having no effect on nucleotide binding to a mutation that apparently abolishes nucleotide binding. One mutation at Asp224 reduced the affinity of beta-tubulin for GTP in the presence but not in the absence of Mg2+. The specific effect of this mutation on nucleotide binding is consistent with an interaction of this amino acid with the Mg2+ moiety of MgGTP. This residue is in a region sharing sequence homology with the putative Mg2+ site in myosin and other ATP-binding proteins. As a result, tubulin belongs to a distinct class of GTP-binding proteins which may be evolutionarily related to the ATP-binding proteins.
J Mol Biol 1992 Sep 05
PMID:Site-directed mutagenesis of the GTP-binding domain of beta-tubulin. 152 95

The crystal structure of trypsin-modified elongation factor Tu from Escherichia coli, in complex with the cofactor guanosine diphosphate has been refined to a crystallographic R-factor of 19.3%, at 2.6 A resolution. In the model described, the root-mean-square deviation from ideality is 0.019 A for bond distances and 3.9 degrees for angles. The protein consists of three domains: an alpha/beta domain (residues 1 to 200), containing the binding site of the GDP cofactor, and consisting of a six-stranded beta-pleated sheet, six alpha-helices, and two all-beta domains (residues 209 to 299 and 300 to 393), belonging to the tertiary structural class of antiparallel beta-barrels. The GDP-binding domain has a folding that is found in other GDP-binding proteins. Elongation factor Tu interacts with proteins, nucleic acids and nucleotides, making this molecule well suited as a model system for the study of these interactions.
J Mol Biol 1992 Feb 05
PMID:Refined structure of elongation factor EF-Tu from Escherichia coli. 154 16

In the plasma membrane of pancreatic B cells, a K+ channel (K-ATP channel) has been identified that is regulated by cytoplasmic nucleotides. This channel is inhibited by sulfonylureas. We have previously shown that the potency of tolbutamide is much lower in excised membrane patches than in intact cells, unless the internal side of the membrane is exposed to the Mg2+ complex of ADP (MgADP). In the present study, the mechanism of this interactive control by sulfonylureas and nucleotides was examined using the inside-out configuration of the patch-clamp technique. When test solutions containing Mg2+ ions were applied, the opening activity of the K-ATP-channels was strongly stimulated by 2'-deoxyadenosine-5'-diphosphate (dADP) or GDP, slightly stimulated by ADP, and inhibited by adenosine-5'-O-(2-thiodiphosphate) (ADP beta S) or adenylyl-imidodiphosphate (AMP-PNP). In the presence of Mg2+, not only ADP but also its analogues dADP (1 mM) and ADP beta S (0.1 mM) enhanced the potency of tolbutamide for channel inhibition; dADP at a low concentration (0.2 mM), GDP (0.2-1 mM), and AMP-PNP (0.2 mM) did not alter the potency of tolbutamide. The particular feature of the test solutions that enhanced the potency of tolbutamide was the presence of Mg(2+)-bound and free nucleotides at channel-stimulating and channel-inhibiting concentrations, respectively. In the presence of Mg2+ and 0.2 mM dADP or 0.2-1 mM GDP, 0.2 mM AMP-PNP intensified the response to tolbutamide by serving as channel-inhibiting component. MgAMP-PNP did not stimulate the opening activity of the K-ATP channel. The sensitivity to tolbutamide that was enhanced by a submaximally effective ADP concentration was further increased by AMP-PNP or ATP but not by GDP. The sensitivity to the sulfonylurea analogue meglitinide was also enhanced by ADP. It is concluded that nucleotides inhibit and activate the K-ATP channel by interaction with two separate receptor sites at the cytoplasmic face of the B cell membrane. Effective inhibition of the channel openings by sulfonylureas results from the simultaneous occupation of both sites by appropriate nucleotides.
Mol Pharmacol 1992 Mar
PMID:Cytosolic nucleotides enhance the tolbutamide sensitivity of the ATP-dependent K+ channel in mouse pancreatic B cells by their combined actions at inhibitory and stimulatory receptors. 154 76

Sec4, a GTP-binding protein of the ras superfamily, is required for exocytosis in the budding yeast Saccharomyces cerevisiae. To test the role of GTP hydrolysis in Sec4 function, we constructed a mutation, Q-79----L, analogous to the oncogenic mutation of Q-61----L in Ras, in a region of Sec4 predicted to interact with the phosphoryl group of GTP. The sec4-leu79 mutation lowers the intrinsic hydrolysis rate to unmeasurable levels. A component of a yeast lysate specifically stimulates the hydrolysis of GTP by Sec4, while the rate of hydrolysis of GTP by Sec4-Leu79 can be stimulated by this GAP activity to only 30% of the stimulated hydrolysis rate of the wild-type protein. The decreased rate of hydrolysis results in the accumulation of the Sec4-Leu79 protein in its GTP-bound form in an overproducing yeast strain. The sec4-leu79 allele can function as the sole copy of sec4 in yeast cells. However, it causes recessive, cold-sensitive growth, a slowing of invertase secretion, and accumulation of secretory vesicles and displays synthetic lethality with a subset of other secretory mutants, indicative of a partial loss of Sec4 function. While the level of Ras function reflects the absolute level of GTP-bound protein, our results suggest that the ability of Sec4 to cycle between its GTP and GDP bound forms is important for its function in vesicular transport, supporting a mechanism for Sec4 function which is distinct from that of the Ras protein.
Mol Cell Biol 1992 May
PMID:Hydrolysis of GTP by Sec4 protein plays an important role in vesicular transport and is stimulated by a GTPase-activating protein in Saccharomyces cerevisiae. 156 38

Genetic data suggest that the yeast cell cycle control gene CDC25 is an upstream regulator of RAS2. We have been able to show for the first time that the guanine nucleotide exchange proteins Cdc25 and Sdc25 from Saccharomyces cerevisiae bind directly to their targets Ras1 and Ras2 in vivo. Using the characteristics of the yeast Ace1 transcriptional activator to probe for protein-protein interaction, we found that the CDC25 gene product binds specifically to wild-type Ras2 but not to the mutated Ras2Val-19 and Ras2 delta Val-19 proteins. The binding properties of Cdc25 to Ras2 were strongly diminished in yeast cells expressing an inactive Ira1 protein, which normally acts as a negative regulator of Ras activity. On the basis of these data, we propose that the ability of Cdc25 to interact with Ras2 proteins is strongly dependent on the activation state of Ras2. Cdc25 binds predominantly to the catalytically inactive GDP-bound form of Ras2, whereas a conformational change of Ras2 to its activated GTP-bound state results in its loss of binding affinity to Cdc25.
Mol Cell Biol 1992 May
PMID:The Saccharomyces cerevisiae CDC25 gene product binds specifically to catalytically inactive ras proteins in vivo. 156 42

Incorporation of the available data on rac in neutrophils, CDC42 in yeast, and rho in fibroblasts suggests a general model for the function of rho-like GTPase (Figure 1). Conversion of an inactive cytoplasmic rho-related p21GDP/GDI complex to active p21. GTP occurs by inhibition of GAP and/or stimulation of exchange factors in response to cell signals. p21.GTP is then able to interact with its target at the plasma membrane. This could result in a conformational change in the target, enabling it to bind cytosolic protein(s). Alternatively, p21.GTP could be actively involved in transporting cytosolic protein(s) to the target. A GAP protein, perhaps intrinsic to the complex, would stimulate GTP hydrolysis allowing p21.GDP to dissociate. Solubilization of p21GDP by interaction with GDI would complete a cycle. What about the nature of the final complex? The rac-regulated NADPH oxidase complex in neutrophils is currently the best understood and most amenable to further biochemical analysis. Two plasma-membrane bound subunits encode the catalytic function necessary for producing superoxide, but the two cytosolic proteins, p47 and p67, are essential for activity. Why the complexity? Production of superoxide is tightly coordinated with phagocytosis, a membrane process driven by rearrangement of cortical actin. This is not unrelated to the membrane ruffling and macropinocytosis that we observe in fibroblasts microinjected with p21rac. It is tempting to speculate, therefore, that in neutrophils rac is involved not only in promoting the assembly of the NADPH oxidase but also in the coordinate reorganization of cortical actin leading to phagocytosis. For CDC42 controlled bud assembly in yeast, the components of the plasma-membrane complex are not so clear. By analogy with rac in neutrophils, it seems likely that CDC42 is involved in promoting the assembly of cytosolic components at the bud site on the plasma membrane. These putative cytosolic proteins have not yet been identified, but BEM1 and ABP1 are two possible candidates. The biochemical basis for the stimulation of adhesion plaques and actin stress fibers by p21rho in fibroblasts is also unclear. However, components of the adhesion plaque such as vinculin and talin are known to be cytosolic when not complexed with integrin receptors, and rho could be involved in regulating their assembly into the adhesion plaque. Several things are still difficult to incorporate into this model. First the target for CDC42, the bud site, although not yet structurally defined requires the activity of another small GTPase, BUD1. Similarly, in activated neutrophils, the NADPH oxidase is found in a complex with rap1, the mammalian homologue of BUD1 (BoKoch et al., 1989). It seems likely, therefore, that the target is not simply a plasma-membrane protein but may be a complex of proteins whose formation is under the control of the rap1/BUD1 GTPase. The other black box in this model is the actin connection: activation of bud assembly by CDC42 is followed by actin polymerization, activation of NADPH oxidase in neutrophils occurs concomitantly with phagocytosis, a cortical actin-dependent process, and p21rho in fibroblasts couples the formation of adhesion plaques to actin stress fibers. One possible link between the GTPase-driven assembly of a plasma-membrane complex and actin polymerization could involve the SH3 domain. Interestingly, both p47 and p67 and yeast ABP1 and BEM1 have SH3 domain. If rho-like GTPases recognize plasma-membrane targets already associated with cortical actin, then this could promote an interaction with a subset of SH3-containing proteins. The result of this would be a GTPase-regulated aggregation of a group of proteins at a single site in the plasma membrane. It is not too difficult to imagine biological processes where such a spatial integration of different biochemical activities would be essential: coupling the assembly of bud components to the formation of actin fibers in yeast; or the activation of NADPH oxidase to phagocytosis in neutrophils; or the assembly of adhesion plaques and the formation of actin stress fibers in fibroblasts are just three examples that have emerged so far. In conclusion, although rho-like GTPases clearly have distinct roles in different mammalian cell types and in yeast, their underlying mechanism of action appears to be strikingly similar. Whether this will remain so when there are some biochemical data to back up these initial observations, time will tell.
Mol Biol Cell 1992 May
PMID:Ras-related GTPases and the cytoskeleton. 161 Nov 53


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