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PubMed1992

Yeast SNF2/SWI2, SNF5, and SNF6 proteins function coordinately with the gene-specific transcriptional activators GAL4 and Bicoid.

The SNF2 (SWI2), SNF5, and SNF6 genes are required for transcription of many diversely regulated genes in Saccharomyces cerevisiae. Previously, we showed that SNF2, SNF5, and SNF6 function interdependently in transcriptional activation, possibly forming a heteromeric complex. Here, we present evidence that SNF6 has a more direct role in stimulating transcription than SNF2 and SNF5. The global effects of mutations in SNF2, SNF5, and SNF6 suggested that these SNF proteins may function coordinately with many gene-specific activators. We show that LexA-GAL4 and LexA-Bicoid fusion proteins are both dependent on SNF2, SNF5, and SNF6 for activation of target genes containing one or multiple lexA operators. The stringency of the requirement for the SNF proteins varies with the activator, the number of binding sites for the activator, and the target promoter. Thus, these SNF proteins appear to represent a class of intermediary proteins that facilitate transcriptional activation by gene-specific regulatory proteins.

Adenosine Triphosphatases↗
PubMed1992

Spatial specificity of chromophore assisted laser inactivation of protein function.

Chromophore assisted laser inactivation (CALI) is a new technique that selectively inactivates proteins of interest to elucidate their in vivo functions. This method has application to a wide array of biological questions. An understanding of aspects of the mechanism of CALI is required for its judicious application. A critical concern for CALI is its spatial specificity because nonspecific inactivation of neighboring unbound proteins by CALI is a possibility. We show here that CALI is very dependent on the distance between the chromophore and the protein such that there is no significant effect beyond 60 A. CALI using antibodies can inactivate other proteins through a complex but its efficacy decreases approximately fourfold for each intervening protein. These data imply that CALI is spatially specific and damage to neighboring proteins is unlikely.

Acetylcholinesterase↗
PubMed1991

Loss of endothelial pertussis toxin-sensitive G protein function in atherosclerotic porcine coronary arteries.

Pertussis toxin, an irreversible inhibitor of some G proteins, inhibits endothelium-dependent relaxations to certain agonists in porcine coronary arteries. In the present study, the effects of the toxin were examined on endothelium-dependent and -independent relaxations of hypercholesterolemic and atherosclerotic porcine coronary arteries to assess the functional state of the endothelial pertussis toxin-sensitive G protein. Male Yorkshire pigs were maintained on either a regular diet (control group, n = 7) or a 2% high-cholesterol diet (cholesterol-fed group, n = 7) for 10 weeks. After the initial 2 weeks of maintenance, animals in both groups underwent balloon catheter removal of the endothelium of the left anterior descending or left circumflex coronary arteries. Endothelium-dependent responses were examined in vitro after 10 weeks of maintenance; at this time, a full lining of endothelial cells in both left coronary arteries was confirmed histologically. In arteries with endothelium of the control group (normal responses), pertussis toxin significantly inhibited the endothelium-dependent relaxations to serotonin, UK14304 (a selective alpha 2-adrenergic receptor agonist), and thrombin but not those to ADP, bradykinin, or the calcium ionophore A23187. In previously denuded arteries of the control group (effects of endothelial regeneration alone) or intact arteries of the cholesterol-fed group (effects of hypercholesterolemia alone), the relaxations to serotonin, UK14304, and thrombin were impaired significantly; those relaxations were impaired further in previously denuded arteries of the cholesterol-fed group (effects of atherosclerosis). The inhibitory effects of pertussis toxin were significantly reduced after endothelial regeneration and in hypercholesterolemia and were almost absent in atherosclerosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1977

The chemical properties of out-of-equilibrium states of proteins and the role of these states in protein functioning.

The out-of-equilibrium states of several iron-containing proteins (cytochromes c of different origin, haemoglobin, myoglobin, ferredoxin and other non-haem iron proteins, cytochrome c oxidase, horseradish peroxidase) were recorded after fast changes in the active centre (electron reduction of iron, ligand dissociation). Strained states result in which the active centre has already been changed and undergone vibrational relaxation but the main part of protein globule is in the 'old', now out-of-equilibrium, state. Protein structure and chemical properties in these states differ considerably from those in equilibrium states. As a rule, the rate constants of protein-specific chemical reactions increase in out-of-equilibrium states by 1--3 orders of magnitude in comparison with those in equilibrium states. Spectra and reactivity of these proteins change in the course of slow (up to 10(-1) s) conformational relaxation, continuously approaching the equilibrium values. It seems that this conformational relaxation is essentially the elementary act of many enzymic reactions for which the rate of substrate-product transformation is determined by the rate of this conformational change.

Cytochrome c Group↗
PubMedDate not supplied

Disturbing GTP-binding protein function through microinjection into the visual cell of Limulus.

We have tested the action of three agents microinjected into the ventral nerve photoreceptor of Limulus on the electrical response to dim light. 1. A monoclonal antibody (mAb 4A) against the G alpha subunit of frog transducin reduces the size of the receptor current to 60%, suggesting an interaction with G alpha in the Limulus photoreceptor. 2. Injection of Clostridium botulinum ADP-ribosyltransferase C3 reduces the size to 46%; latency is not affected. The results imply that small GTP-binding proteins play a functional role in photoreception of invertebrates. 3. Injection of GDP-beta-S reduces dose-dependently the size of the receptor current to 15% and prolongs the latency to 200%, presumably by reducing number and rate of G-protein activations.

Animals↗
PubMed1990

Cloning of ubiquitin activating enzyme from wheat and expression of a functional protein in Escherichia coli.

The initial step in the conjugation of ubiquitin to substrate proteins involves the activation of ubiquitin by ubiquitin activating enzyme, E1. Previously, we purified and characterized multiple species of E1 from wheat germ. We now describe the isolation and characterization of a cDNA clone encoding E1 from wheat. This clone (UBA1) was isolated from a cDNA expression library with anti-wheat E1 antibodies. It contained an open reading frame coding for 1051 amino acids and directed the synthesis of a protein that comigrated with a wheat germ E1 of 117 kDa. UBA1 was confirmed as encoding E1 by (i) comparison of the peptide map of the protein product of UBA1 synthesized in Escherichia coli with that of purified E1 from wheat, and (ii) amino acid sequence identity of peptides generated from purified E1 with regions of the derived amino acid sequence of UBA1. The isolation of two additional cDNAs closely related to UBA1 indicated that E1 was encoded by a small gene family in wheat. Nonetheless, a single poly(A+) mRNA size class of 4 kilobases hybridized with UBA1. When expressed in E. coli, the product of UBA1 catalyzed the formation of a thiol ester linkage between ubiquitin and an ubiquitin carrier protein. The ability of E. coli containing UBA1 to synthesize an active protein will allow us to identify domains important for E1 function using in vitro mutagenesis.

Amino Acid Sequence↗
PubMed1991

Cholinesterase-like domains in enzymes and structural proteins: functional and evolutionary relationships and identification of a catalytically essential aspartic acid.

Primary sequences of cholinesterases and related proteins have been systematically compared. The cholinesterase-like domain of these proteins, about 500 amino acids, may fulfill a catalytic and a structural function. We identified an aspartic acid residue that is conserved among esterases and lipases (Asp-397 in Torpedo acetylcholinesterase) but that had not been considered to be involved in the catalytic mechanism. Site-directed mutagenesis demonstrated that this residue is necessary for activity. Analysis of evolutionary relationships shows that the noncatalytic members of the family do not constitute a separate subgroup, suggesting that loss of catalytic activity occurred independently on several occasions, probably from bifunctional molecules. Cholinesterases may thus be involved in cell-cell interactions in addition to the hydrolysis of acetylcholine. This would explain their specific expression in well-defined territories during embryogenesis before the formation of cholinergic synapses and their presence in noncholinergic tissues.

Acetylcholinesterase↗
PubMed1990

Tropoelastin heterogeneity: implications for protein function and disease.

The organization of the tropoelastin gene is similar to that of other genes coding for matrix proteins in that the exons code for distinct domains of the protein. An unusual feature of tropoelastin expression is that the primary transcript of the gene coding for tropoelastin undergoes extensive, developmentally regulated alternative splicing, resulting in numerous protein isoforms. Although the significance of this heterogeneity is unknown, the multiple sequence variations may affect the function of tropoelastin. Without an understanding of the importance of the domains of tropoelastin and the process of fibrillogenesis, characterization of defects resulting in aberrant elastin production will be hindered. In this update, we review recent findings on tropoelastin and speculate as to the structural and regulatory role of various regions of this matrix protein.

Amino Acid Sequence↗
PubMed1991

Silencer binding proteins function on multiple cis-elements in the glutathione transferase P gene.

The glutathione transferase P (GST-P) gene is specifically expressed during chemical hepatocarcinogenesis of the rat, whereas mRNA of this gene is virtually undetectable in normal liver. We have previously identified a stretch of DNA, that acted negatively in transcription, at 400 bp upstream from the cap site of the rat GST-P gene. Further characterization has revealed that this negative fragment functions in an orientation and position independent manner, suggesting that it is acting as a silencer. This silencer consists of multiple negative elements to which nuclear factors bind. This silencer is active not only in rat non-hepatoma and hepatoma cells but also in human and mouse cell lines, suggesting that these elements function as general regulators of basal gene expression. At least two proteins bind to this silencer fragment, one of which, designated SF-A (Silencer Factor A), has been partially purified. SF-A binds to several regions in this silencer, and likely plays an important role on negative regulation of this gene.

Animals↗
PubMed1990

[Effects of ethanol on GTP binding protein function in rat cerebral cortex: strain difference between Wistar and Fischer 344 rats].

The effect of ethanol on GTP-binding protein of cortical adenylate cyclase (AC) system in the Wistar and Fischer 344 rats was examined. In both Wistar and Fischer 344 rats, ethanol (100mM) potentiated Gpp (NH) p-stimulated AC activity and decreased EC50 for Gpp (NH)p in the cerebral cortical membrane (CCM). Ethanol treatment also augmented Mn-stimulated AC activity in Wistar CCM, whereas there was no significant change in Fischer 344 CCM. IC50 calculated from the Gpp (NH)p (0.01-0.3 microM) inhibition of forskolin-stimulated AC was decreased by ethanol treatment in Wistar CCM, but this was not the case in Fischer 344 CCM. There was no appreciable change in ADP-ribosylation of Gs and Gi in CCM with pertussis or cholera toxin. These results suggest that ethanol in vitro enhances the Gi function as well as affecting the Gs function in Wistar rat cortical AC system, and the effect of ethanol on Gi protein are different in Wistar and Fischer 344 CCM.

Adenylyl Cyclases↗
PubMed1991

Reconstitution of GTP-binding Sar1 protein function in ER to Golgi transport.

In the yeast secretory pathway, two genes SEC12 and SAR1, which encode a 70-kD integral membrane protein and a 21-kD GTP-binding protein, respectively, cooperate in protein transport from the ER to the Golgi apparatus. In vivo, the elevation of the SAR1 dosage suppresses temperature sensitivity of the sec12 mutant. In this paper, we show cell-free reconstitution of the ER-to-Golgi transport that depends on both of these gene products. First, the membranes from the sec12 mutant cells reproduce temperature sensitivity in the in vitro ER-to-Golgi transport reaction. Furthermore, the addition of the Sar1 protein completely suppresses this temperature-sensitive defect of the sec12 membranes. The analysis of Sar1p partially purified by E. coli expression suggests that GTP hydrolysis is essential for Sar1p to execute its function.

Cloning, Molecular↗
PubMed1992

Carboxyl-terminal consensus Ser-Lys-Leu-related tripeptide of peroxisomal proteins functions in vitro as a minimal peroxisome-targeting signal.

The minimal sequence requirement for a peroxisome-targeting signal was investigated using an in vitro import system. Carboxyl-terminal sequences Ser-Lys-Leu (SKL) and Leu-Gln-Ser-Lys-Leu (LQSKL) of acyl-CoA oxidase (AOX) directed to peroxisomes the fused proteins with import-incompetent forms of AOX and catalase that had been truncated, implying that the SKL tripeptide functions as a targeting signal. Elimination of the entire SKL sequence or deletion of any 1 or 2 amino acids in the sequence abolished the import activity of AOX. Substitution of alanine for serine did not affect the import activity. Topogenic activity was retained when lysine was mutated to either arginine or histidine, whereas mutation to glutamic acid completely abolished the activity. A synthetic peptide comprising the carboxyl-terminal 10 amino acid residues of AOX inhibited the import of the authentic AOX polypeptide, whereas other peptides in which SKL was mutated, deleted, or internally located were not effective. The uptake of AOX was little affected by the peptide with an amidated alpha-carboxyl group. These results strongly suggest that the carboxyl-terminal SKL motif sequence (Ser/Ala)-(Lys/Arg/His)-Leu functions as a topogenic signal in translocation of proteins into peroxisomes, requiring the whole tripeptide sequence with a free alpha-COOH group at the carboxyl terminus.

Acyl-CoA Oxidase↗
PubMed1976

Renal function, protein binding and pharmacological response to diazoxide.

1 The effect of rapid (10s) injections of diazoxide was studied in ten hypertensive patients with varying degrees of impairment of renal function. 2 There was a significant correlation between the patient's plasma urea concentration and reduction in mean arterial blood pressure. Diazoxide was also shown to be less highly protein bound in patients with renal failure. 3 It is suggested that the explanation for the increased hypotensive effect of diazoxide observed in patients with reduced renal function is related to higher unbound drug concentrations.

Adult↗
PubMed1990

Alternatively spliced transcripts of the sex-determining gene tra-2 of Drosophila encode functional proteins of different size.

The Drosophila transformer-2 gene (tra-2) is required for female sex determination in somatic cells and for spermatogenesis in male germ cells. We studied the organization of the tra-2 gene and characterized the transcripts in wild type and mutant animals. Two transcripts are detected in males and females; they differ in their abundance and in the presence (minor transcript Tmin) or absence (major transcript Tmaj) of one exon. Two other transcripts are present only in male germ cells. One of these is rare (msTmin) and represents a spliced form of the other, more abundant transcript (msTmaj). The transcript Tmaj encodes a protein of 264 amino acids, whereas transcripts Tmin and msTmaj encode proteins that are truncated at the N-terminus. All three putative proteins contain a stretch of approximately 90 amino acids, the ribonucleoprotein motif (RNP motif), which shows similarity to a variety of different ribonucleoproteins. Transformation studies reveal that a cDNA corresponding to the transcript Tmaj can provide all the functions for female sex determination and male fertility. Surprisingly, a cDNA corresponding to the transcript msTmaj could only supply some female sex-determining function, but was unable to restore fertility in mutant males. Sequence analysis of two temperature-sensitive mutations provides evidence that the RNP motif represents an important functional domain of the tra-2 protein.

Amino Acid Sequence↗