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Results for “Functional characterization”

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At least 649 records · Page 36Linked to original sources

PubMed2005

Identification, expression and functional characterization of the GRAL gene.

The glial cell line-derived neurotrophic factor (GDNF) family is a group of neurotrophic factors with diverse biological functions. Members of the GDNF family exert their functions by interacting with a specific GDNF family receptor alpha (GFRalpha) and activation of the cRET. Here we report the identification and characterization of GDNF receptor-alpha-like (GRAL) gene. Sequence analysis indicated that GRAL is a distant homolog of the GFRalpha family, with 30% of its amino acid sequence identical to that of GFRalpha-3. There are two splice variants of GRAL: the full-length form (GRAL-A) represented by a 2080 bp mRNA and a short form (GRAL-B) represented by a 1833 bp mRNA. In adult mouse, GRAL transcripts have been found primarily in the CNS. In the developing mouse brain, the mRNA level of GRAL in the cerebrocortex and hippocampus reached a maximum at birth and declined afterwards. GRAL-A protein was localized predominantly in the plasma membrane. Overexpression of GRAL-A protected PC12 cells and cultured hippocampal neurons from serum starvation-induced cell apoptosis. The neuroprotective effect of GRAL was associated with marked inhibition of the Jun-N-terminal kinase signaling pathway. Our results suggest that GRAL belongs to a superfamily of GFRalpha and might take part in neuroprotection and brain development.

Amino Acid Sequence↗

PubMed2004

Molecular and functional characterization of proteins interacting with the C-terminal domains of 5-HT2 receptors: emergence of 5-HT2 "receptosomes".

Many cellular functions are carried out by multiprotein complexes. The last five years of research have revealed that many G-protein coupled receptor (GPCR) functions that are not mediated by G proteins involve protein networks, which interact with their intracellular domains. This review focuses on one family of GPCRs activated by serotonin, the 5-HT(2) receptor family, which comprises three closely related subtypes, the 5-HT(2A), the 5-HT(2B) and the 5-HT(2c) receptors. These receptors still raise particular interest, because a large number of psychoactive drugs including hallucinogens, anti-psychotics, anxiolytics and anti-depressants, mediate their action, at least in part, through activation of 5-HT(2) receptors. Recent studies based on two-hybrid screens, proteomic, biochemical and cell biology approaches, have shown that the C-terminal domains of 5-HT(2) receptors interact with intracellular proteins. To date, the protein network associated with the C-terminus of the 5-HT(2C) receptor has been the most extensively characterized, using a proteomic approach combining affinity chromatography, mass spectrometry and immunoblotting. It includes scaffolding proteins containing one or several PDZ domains, signalling proteins and proteins of the cytoskeleton. Data indicating that the protein complexes interacting with 5-HT(2) receptor C-termini tightly control receptor trafficking and receptor-mediated signalling will also be reviewed.

Animals↗

PubMed1996

Biochemical and functional characterization of basal lamina-bound agrin in the chick central nervous system.

Agrin is a high-molecular weight extracellular matrix molecule, initially purified from the electric organ of the marine ray Torpedo californica, which induces on the surface of cultured myotubes the formation of postsynaptic specializations similar to those found at the neuromuscular junction. Agrin immunoreactivity is highly concentrated in the basal lamina of the synaptic cleft but is also found in a number of other tissues where its function is not known. We characterized agrin associated with two basal laminae from the central nervous system, the inner limiting membrane of the retina and the mesencephalic external limiting membrane. A major broad band with an apparent molecular weight of > 300 kDa was identified in immunoblots of isolated basal laminae from retina, mesencephalon, kidney and muscle, showing that basal lamina-bound agrin from the central nervous system and that from non-neural tissues have similar molecular sizes. Agrin is stably but not covalently bound to the inner limiting membrane and could be completely removed only with strong detergents. Agrin could be partially extracted with buffers that are also able to partially release acetylcholine receptor aggregation activity from the neuromuscular junction or from the electric organ. Despite these immunological and biochemical similarities, agrin from both central nervous system-derived basal laminae was not able to induce acetylcholine receptor aggregation on cultured myotubes. This shows that functionally different agrin isoforms are associated with basal laminae in the central nervous system compared to the neuromuscular junction or the electric organ.

Agrin↗

PubMed2004

Expression and functional characterization of the system L amino acid transporter in KB human oral epidermoid carcinoma cells.

We have examined the expression and function of system l amino acid transporter in KB human oral epidermoid carcinoma cells. The KB cells express l-type amino acid transporter 1 (LAT1) in plasma membrane, but not l-type amino acid transporter 2 (LAT2). The [14C]l-leucine uptake by KB cells is inhibited by system l selective inhibitor BCH. The majority of [14C]l-leucine uptake is, therefore, mediated by LAT1. These results suggest that the transport of neutral amino acids including several essential amino acids into the KB cells mediated by LAT1 and the specific inhibition of LAT1 in oral cancer cells will be a new rationale for anti-cancer therapy.

Amino Acid Transport System y+↗

PubMed2002

Expression of ten RGS proteins in human myocardium: functional characterization of an upregulation of RGS4 in heart failure.

OBJECTIVE: RGS proteins (regulators of G protein signalling) negatively regulate G protein function as GTPase activating proteins. By controlling heterotrimeric G proteins they may regulate myocardial hypertrophy and contractility. We investigated the expression of RGS proteins in the human heart and whether they take part in the pathophysiological changes of heart failure. METHODS AND RESULTS: Using RNase protection assays (RPAs) RGS2, 3L, 3S, 4, 5 and 6 were identified in the myocardium from terminally failing human hearts with dilated (DCM, n=22) or ischemic (ICM, n=18) cardiomyopathy and from nonfailing donor hearts (NF, n=9). With reverse transcriptase polymerase chain reaction in addition mRNA of RGS1, 9, 12, 14 and 16 were detectable. Compared to NF in failing LV myocardium RGS4 mRNA and protein was upregulated 2-3-fold (mRNA, 10(-21) mol/microg+/-S.E.M.: NF: 22+/-5, DCM: 51+/-10*, ICM: 37+/-8; P<0.05 vs. DCM+ICM, *P<0.05 vs. NF, P<0.05 vs. DCM+ICM; protein, % of NF+/-S.E.M.: NF: 100+/-35, DCM 266+/-60*, ICM: 205+/-64, n=5, *P<0.05 vs. NF). In contrast, RGS2, 3L, 3S, 5, 6, and 16 protein and mRNA levels did not vary between failing and NF hearts. In order to investigate the impact of RGS4 on Gq/11 mediated signalling, PLC activity was measured in human LV membranes. Recombinant RGS4 blunted the endothelin-1 (ET-1) stimulated PLC activity. When overexpressed by adenoviral mediated gene transfer in rabbit ventricular myocytes RGS4 abolished the inotropic effect of ET-1. CONCLUSION: The upregulation of RGS4 in failing human myocardium diminishes Gq/11-mediated signalling and can be involved in the desensitization of Gq/11-mediated positive inotropic effects.

Animals↗

PubMed1998

Sequence and functional characterization of RNase P RNA from the chl alb containing cyanobacterium Prochlorothrix hollandica.

Only a few complete sequences and very limited functional data are available for the catalytic RNA component of cyanobacterial RNase P. The RNase P RNA from the chl alb containing cyanobacterium Prochlorothrix hollandica belongs to a rarely found structural subtype with an extended P15/16 domain. We have established conditions for optimal in vitro ribozyme activity, and determined the kinetic parameters for cleavage of pre-tRNA(Tyr). Analysis of pre-tRNA mutants revealed that the T-stem sequence only plays a modulating role, whereas the CCA end is essential for efficient product formation.

Base Sequence↗

PubMed2004

Distant structural homology leads to the functional characterization of an archaeal PIN domain as an exonuclease.

Genome sequencing projects have focused attention on the problem of discovering the functions of protein domains that are widely distributed throughout living species but which are, as yet, largely uncharacterized. One such example is the PIN domain, found in eukaryotes, bacteria, and Archaea, and with suggested roles in signaling, RNase editing, and/or nucleotide binding. The first reported crystal structure of a PIN domain (open reading frame PAE2754, derived from the crenarchaeon, Pyrobaculum aerophilum) has been determined to 2.5 A resolution and is presented here. Mapping conserved residues from a multiple sequence alignment onto the structure identifies a putative active site. The discovery of distant structural homology with several exonucleases, including T4 phage RNase H and flap endonuclease (FEN1), further suggests a likely function for PIN domains as Mg2+-dependent exonucleases, a hypothesis that we have confirmed in vitro. The tetrameric structure of PAE2754, with the active sites inside a tunnel, suggests a mechanism for selective cleavage of single-stranded overhangs or flap structures. These results indicate likely DNA or RNA editing roles for prokaryotic PIN domains, which are strikingly numerous in thermophiles, and in organisms such as Mycobacterium tuberculosis. They also support previous hypotheses that eukaryotic PIN domains participate in RNAi and nonsense-mediated RNA degradation.

Amino Acid Sequence↗

PubMed2006

Functional characterization of a fatty acyl-CoA-binding protein (ACBP) from the apicomplexan Cryptosporidium parvum.

In this paper, the identification and functional analysis of a fatty acyl-CoA-binding protein (ACBP) gene from the opportunistic protist Cryptosporidium parvum are described. The CpACBP1 gene encodes a protein of 268 aa that is three times larger than typical ACBPs (i.e. approximately 90 aa) of humans and animals. Sequence analysis indicated that the CpACBP1 protein consists of an N-terminal ACBP domain (approximately 90 aa) and a C-terminal ankyrin repeat sequence (approximately 170 aa). The entire CpACBP1 ORF was engineered into a maltose-binding protein fusion system and expressed as a recombinant protein for functional analysis. Acyl-CoA-binding assays clearly revealed that the preferred binding substrate for CpACBP1 is palmitoyl-CoA. RT-PCR, Western blotting and immunolabelling analyses clearly showed that the CpACBP1 gene is mainly expressed during the intracellular developmental stages and that the level increases during parasite development. Immunofluorescence microscopy showed that CpACBP1 is associated with the parasitophorous vacuole membrane (PVM), which implies that this protein may be involved in lipid remodelling in the PVM, or in the transport of fatty acids across the membrane.

Acyl Coenzyme A↗

PubMed1993

Molecular cloning, expression and functional characterization of rabbit anticoagulant vitamin-K-dependent protein S.

Vitamin-K-dependent protein S is an anticoagulant plasma protein which functions as cofactor to activated protein C (APC) in the degradation of coagulation factors Va and VIIIa. In addition, it interacts with C4b-binding protein (C4BP), a regulator of the complement system. Using a human protein S cDNA clone as probe, cDNA clones for rabbit protein S were isolated from a rabbit liver cDNA library. The cDNA sequence encoded the mature protein S and 12 residues of the leader sequence. The amino acid sequence of the single-chain 634-amino-acid-residue-long rabbit protein S molecule was 82% and 81% identical to those of human and bovine protein S, respectively. Northern blotting demonstrated protein S mRNA not only in liver but also in reproductive organs (testis, ovary and uterus), in lung and brain. Recombinant rabbit protein S was expressed in eucaryotic cells and found to be post-translationally modified, i.e. it had the correct amino terminus, contained N-linked carbohydrate side chains, gamma-carboxyglutamic acid residues and beta-hydroxylated aspartic acid and asparagine residues. Recombinant rabbit protein S bound calcium like its human counterpart, as judged by its migration in the presence of calcium on agarose-gel electrophoresis. Rabbit protein S has been reported to be species specific with respect to its interaction with APC and not to function with bovine APC. However, we found it to act as cofactor to both human and bovine APC, albeit it was somewhat more efficient with human than with bovine APC. Rabbit protein S, like its human and bovine counterparts, bound human C4BP in a reaction which was associated with the loss of its APC-cofactor activity. However, unlike human plasma, rabbit plasma appeared to contain only the free form of protein S as a radiolabeled rabbit protein S tracer added to rabbit plasma migrated as free protein S on agarose-gel electrophoresis. Addition of human C4BP to rabbit plasma resulted in the formation of a C4BP-protein-S complex, suggesting an explanation for the absence of complexed protein S in rabbit plasma to be sought for in the structure of rabbit C4BP.

Amino Acid Sequence↗

PubMed2000

Functional characterization of the human immunodeficiency virus type 1 genome by genetic footprinting.

We present a detailed and quantitative analysis of the functional characteristics of the 1,000-nucleotide segment at the 5' end of the human immunodeficiency virus type 1 (HIV-1) RNA genome. This segment of the viral genome contains several important cis-acting sequences, including the TAR, polyadenylation, viral att site, minus-strand primer-binding site, and 5' splice donor sequences, as well as coding sequences for the matrix protein and the N-terminal half of the capsid protein. The genetic footprinting technique was used to determine quantitatively the abilities of 134 independent insertion mutations to (i) make stable viral RNA, (ii) assemble and release viral RNA-containing viral particles, and (iii) enter host cells, complete reverse transcription, enter the nuclei of host cells, and generate proviruses in the host genome by integration. All of the mutants were constructed and analyzed en masse, greatly decreasing the labor typically involved in mutagenesis studies. The results confirmed the presence of several previously known functional features in this region of the HIV genome and provided evidence for several novel features, including newly identified cis-acting sequences that appeared to contribute to (i) the formation of stable viral transcripts, (ii) viral RNA packaging, and (iii) an early step in viral replication. The results also pointed to an unanticipated trans-acting role for the N-terminal portion of matrix in the formation of stable viral RNA transcripts. Finally, in contrast to previous reports, the results of this study suggested that detrimental mutations in the matrix and capsid proteins principally interfered with viral assembly.

Cell Line, Transformed↗

PubMed2003

Functional characterization of bacterial signal peptide OmpA in a baculovirus-mediated expression system.

Signal sequences are evolutionarily conserved and are often functionally interchangeable between prokaryotes and eukaryotes. However, we have found that the bacterial signal peptide, OmpA, functions incompletely in insect cells. Upon baculovirus-mediated expression of chloramphenicol acetyltransferase (CAT) in insect cells, OmpA signal peptide led to the cytosolic accumulation of the CAT molecules in an aglycosylated, signal-peptide cleaved form, in addition to the secretion of the glycosylated CAT. When green fluorescent protein (GFP) was used as another reporter, the GFP molecules expressed from the OmpA-GFP construct was distributed primarily in the cytosol as aggresome-like structures. These results together suggest that, subsequent to the cleavage of OmpA signal peptide in the ER, some of the processed proteins are returned to the cytoplasm. Since the prototypical insect signal peptide, melittin, did not result in this ER-to-cytosol dislocation of the reporter proteins, we proposed a model explaining the dislocation process in insect cells, apparently selective to the OmpA-directed secretory pathway bypassing the co-translational transport.

Animals↗

PubMed1997

The nucleoside-specific Tsx channel from the outer membrane of Salmonella typhimurium, Klebsiella pneumoniae and Enterobacter aerogenes: functional characterization and DNA sequence analysis of the tsx genes.

The Escherichia coli tsx gene encodes an integral outer-membrane protein (Tsx) that functions as a substrate-specific channel for deoxynucleosides and the antibiotic albicidin, and also serves as a receptor for bacteriophages and colicins. We cloned the structural genes of the Tsx proteins from Salmonella typhimurium, Klebsiella pneumoniae and Enterobacter aerogenes and expressed them in an E.coli tsx mutant. The heterologous Tsx proteins fully substituted the E.coli Tsx protein with respect to its function in deoxynucleoside and albicidin uptake, and as receptor for colicin K. The Tsx proteins from K. pneumoniae and Ent. aerogenes were also proficient as receptors for several Tsx-specific bacteriophages, whereas the corresponding protein from S. typhimurium did not confer sensitivity against these phages. The nucleotide sequence of the tsx genes from S. typhimurium, K. pneumoniae and Ent. aerogenes was established. Each of the Tsx proteins is initially synthesized with typical bacterial signal sequence peptides and the predicted mature forms of the Tsx proteins have a calculated M(r) of 30,567 (265 residues), 31,412 (272 residues) and 31,477 (272 residues), respectively. Multiple sequence alignments between the Tsx proteins showed a high degree of sequence identity and revealed the presence of four hypervariable regions, which are thought to constitute segments of the polypeptide chain exposed at the cell surface. Most notable was a deletion of 8 amino acids in one of these hypervariable domains in the S. typhimurium Tsx protein. When this deletion was introduced by site-directed mutagenesis into the corresponding region of the E.coli tsx gene, the mutant Tsx-515 protein lost its phage receptor function but still served as a colicin K receptor and as a substrate-specific channel, indicating that the region between residues 198 and 207 might be part of the bacteriophage receptor area. Multiple sequence alignments, structural predictions and the properties of previously characterized Tsx missense mutants were taken into account to develop a two-dimensional model for the topological organization of the Tsx protein within the outer membrane.

Amino Acid Sequence↗

PubMed2006

Phenotypical and functional characterization of alveolar macrophage subpopulations in the lungs of NO2-exposed rats.

BACKGROUND: Alveolar macrophages (AM) are known to play an important role in the regulation of inflammatory reactions in the lung, e.g. during the development of chronic lung diseases. Exposure of rats to NO2 has recently been shown to induce a shift in the activation type of AM that is characterized by reduced TNF-alpha and increased IL-10 production. So far it is unclear, whether a functional shift in the already present AM population or the occurrence of a new, phenotypically different AM population is responsible for these observations. METHODS: AM from rat and mice were analyzed by flow cytometry for surface marker expression and in vivo staining with PKH26 was applied to characterize newly recruited macrophages. Following magnetic bead separation, AM subpopulations were further analyzed for cytokine, inducible NO synthase (iNOS) and matrix metalloproteinase (MMP) mRNA expression using quantitative RT-PCR. Following in vitro stimulation, cytokines were quantitated in the culture supernatants by ELISA. RESULTS: In untreated rats the majority of AM showed a low expression of the surface antigen ED7 (CD11b) and a high ED9 (CD172) expression (ED7-/ED9high). In contrast, NO2 exposure induced the occurrence of a subpopulation characterized by the marker combination ED7+/ED9low. Comparable changes were observed in mice and by in vivo labeling of resident AM using the dye PKH26 we could demonstrate that CD11b positive cells mainly comprise newly recruited AM. Subsequent functional analyses of separated AM subpopulations of the rat revealed that ED7+ cells showed an increased expression and production of the antiinflammatory cytokine IL-10 whereas TNF-alpha production was lower compared to ED7- AM. However, iNOS and IL-12 expression were also increased in the ED7+ subpopulation. In addition, these cells showed a significantly higher mRNA expression for the matrix metalloproteinases MMP-7, -8, -9, and -12. CONCLUSION: NO2 exposure induces the infiltration of an AM subpopulation that, on the one hand may exert antiinflammatory functions by the production of high amounts of IL-10 but on the other hand may contribute to the pathology of NO2-induced lung damage by selective expression of certain matrix metalloproteinases.

Animals↗

PubMed2003

Cloning and functional characterization of GMPR2, a novel human guanosine monophosphate reductase, which promotes the monocytic differentiation of HL-60 leukemia cells.

PURPOSE: To identify the biological function of a novel molecule which shows high homology with human guanosine monophosphate reductase (GMPR) and is named GMPR2. METHODS: GMPR2 cDNA was cloned from the cDNA library of human dendritic cells and was characterized by Bioinformatics. The expression pattern of GMPR2 was analyzed by Northern blotting. The enzymatic activity of the purified recombinant GMPR2 protein was determined using a spectrophotometric assay. HL-60 leukemia cells were transfected with GMPR2 and the expression of CD14 and myeloperoxidase (MPO) in HL-60 cells with and without 12- o-tetra-decanoyl-phorbol-13-acetate (TPA) induction was monitored by FACS analysis. RESULTS: The novel gene contained ten exons and nine introns and was mapped to 14q11-21. Northern blotting indicated a ubiquitous expression of GMPR2 mRNA in most of the human tissues and cancer cell lines investigated. The recombinant GMPR2 protein was able to reduce GMP. The expression of CD14 and MPO in HL-60 leukemia cells overexpressing GMPR2 clearly increased after induction by TPA. CONCLUSIONS: GMPR2 is a novel human GMP reductase, and overexpression of GMPR2 can promote the monocytic differentiation of HL-60 leukemia cells.

Amino Acid Sequence↗

PubMed2004

Radioligand binding and functional characterization of recombinant human NmU1 and NmU2 receptors stably expressed in clonal human embryonic kidney-293 cells.

Neuromedin U (NmU) is a smooth muscle contracting peptide. Recently, two G-protein-coupled receptors for NmU (NmU1R and NmU2R) have been cloned having approximately 50% homology. They have distinct patterns of expression suggesting they may have different biological functions. This study provides a comprehensive characterization of both NmU receptors expressed in human embryonic kidney 293 cells. [125I]hNmU binding to the recombinant NmU receptors was rapid, saturable, of high affinity and to a single population of binding sites. Exposure of these cells to NmU isopeptides resulted in an increase in intracellular [Ca2+]i release (EC50 value of 0.50 +/- 0.10 nmol/l) and inositol phosphate formation (EC50 1.6 +/- 0.2 and 1.50 +/- 0.4 nmol/l for NmU1R and NmU2R respectively). Furthermore, hNmU inhibited forskolin (3 micromol/l)-stimulated accumulation of cAMP in intact HEK-293 cells expressing either NmU1R or NmU2R. The inhibitory effect was significant for the cells expressing NmU2R with IC50 value of 0.80 +/- 0.21 nmol/l. In summary, both NmU1R and NmU2R in HEK-293 cells have similar signaling capability.

Binding Sites↗

PubMed2005

Heterologous expression of piglet odorant-binding protein in Pichia pastoris: a comparative structural and functional characterization with native forms.

This study targets to express the piglet odorant-binding protein (plOBP) and compare the engineered product to the corresponding native protein forms, i.e. plOBP and adult porcine OBP (pOBP). Using the natural signal peptide from the cDNA sequence, up to 40 mg l(-1) of secreted recombinant piglet OBP (rOBP) has been produced in a minimal culture medium. No significant difference in molecular mass between rOBP and native plOBP could be observed by mass spectrometry following or not trypsin digestion. rOBP and pOBP shared similar immunoreactivity towards polyclonal anti-pOBP antibodies, suggesting a proper processing and folding of the recombinant product. Both plOBP and rOBP displayed comparable binding properties towards fatty acids present in the putative maternal pheromone and a steroid, component of the boar sex pheromone. Furthermore, the rOBP product was found to bind to an olfactory receptor, for which pOBP binding was previously characterized. Taken together, these findings suggest that rOBP, produced in Pichia pastoris, exhibits structural and functional properties comparable to those of the native lipocalins from both young or adult animal.

Animals↗

PubMed2005

Structural and functional characterization of Mycobacterium tuberculosis CmtR, a PbII/CdII-sensing SmtB/ArsR metalloregulatory repressor.

The SmtB/ArsR family of prokaryotic metalloregulators are winged-helix transcriptional repressors that collectively provide resistance to a wide range of both biologically required and toxic heavy-metal ions. CmtR is a recently described Cd(II)/Pb(II) regulator expressed in Mycobacterium tuberculosis that is structurally distinct from the well-characterized SmtB/ArsR Cd(II)/Pb(II) sensor, Staphylococcus aureus plasmid pI258-encoded CadC. From functional analyses and a multiple sequence alignment of CmtR paralogs, M. tuberculosis CmtR is proposed to bind Pb(II) and Cd(II) via coordination by Cys57, Cys61, and Cys102 [Cavet et al. (2003) J. Biol. Chem. 278, 44560-44566]. We establish here that both wild-type and C102S CmtR are homodimers and bind Cd(II) and Pb(II) via formation of cysteine thiolate-rich coordination bonds. UV-vis optical spectroscopy, (113)Cd NMR spectroscopy (delta = 480 ppm), and (111m)Cd perturbed angular correlation (PAC) spectroscopy suggest two or three thiolate donors in the wild-type protein. Cys57 and Cys61 anchor the coordination complex, while Cys102 plays only an accessory role in stabilizing the metal chelate in the free protein because C102S CmtR binds Cd(II) and Zn(II) with only approximately 10-20-fold lower affinity relative to wild-type CmtR but approximately 100-1000-fold lower for Pb(II). Quantitative investigation of CmtR-cmt O/P binding equilibria using fluorescence anisotropy, however, reveals that Cys102 functions as a key allosteric metal ligand, because substitution of Cys102 abrogates disassembly of oligomeric CmtR-cmt O/P oligomeric complexes. The implications of these findings on the evolution of distinct metal-sensing sites in a family of homologous proteins are discussed.

Amino Acid Sequence↗

PubMed1992

Functional characterization of temperature-sensitive mutants of simian virus 40 large T antigen.

We investigated the molecular properties of eight temperature-sensitive mutants of simian virus 40 large T antigen (tsA mutants). The mutants have single amino acid substitutions that block DNA replication at 39 to 41 degrees C in vivo. In vitro, five of the mutant proteins were highly sensitive to a brief heat shock at 39 degrees C, while the three remaining proteins were only partially sensitive at 41 degrees C. We characterized the five most defective mutant proteins, using a variety of biochemical assays for replication functions of T antigen. Heat shock of purified T antigen with a mutation at amino acid 422 significantly impaired the oligomerization, origin-binding, origin-unwinding, ATPase, and helicase functions of T antigen. In contrast, substitution of amino acid 186, 357, 427, or 438 had more selective, temperature-sensitive effects on T-antigen functions. Our findings are consistent with the conclusion that T antigen functions via a hierarchy of interrelated domains. Only the ATPase activity remained intact in the absence of all other functions. Hexamer formation appears to be necessary for core origin-unwinding and helicase activities; the helicase function also requires ATPase activity. All five tsA mutants were impaired in functions important for the initiation of DNA replication, but three mutants retained significant elongation functions.

Adenosine Triphosphatases↗