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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↗
PubMed1995

Gene-targeting and transgenic approaches to IGF and IGF binding protein function.

The ability to manipulate genetic information in the germ line of mice has provided powerful approaches to study gene function in vivo. These approaches have included the establishment of mouse lines in which a specified gene or genes are overexpressed, ectopically expressed, or deleted. Transgenic and gene-targeted mouse lines have been used extensively to study the function of the insulin-like growth factors (IGF), IGF-I and IGF-II, and their receptors and binding proteins. In the IGF system, these technologies have elucidated the roles of the IGFs in fetal and somatic growth and have demonstrated a critical role for this system in transformation and tumorigenesis. Analysis of combinatorial crosses of gene-targeted mouse lines also has suggested the existence of an as yet unidentified IGF receptor that regulates fetal growth. Similar approaches using transgenic and gene-targeted mouse models have been initiated to study the in vivo functions of the IGF binding proteins. These mouse models provide important tools to test specific functional questions in vivo as well as to study the long-term physiological consequences of chronic gene alterations.

Animals↗
PubMed1993

Pathogenic and non-pathogenic T lymphocytes specific for the encephalitogenic epitope of myelin basic protein: functional characteristics and vaccination properties.

Activated CD4+ T lymphocytes specific for myelin basic protein (MBP) can cause experimental autoimmune encephalomyelitis (EAE) upon their inoculation into syngeneic recipients. In Lewis rats, most of the pathogenic T cell clones that develop following immunization with MBP are reactive against the 72-84 amino acid sequence of MBP, the major encephalitogenic region for Lewis rats. In this study, some MBP-specific T cell clones were found to be non-pathogenic, in spite of their strong reactivity against the encephalitogenic epitope. One of these non-pathogenic clones, designated Znp, and an encephalitogenic clone, Z1a-p, were derived from Z1a encephalitogenic line cells. These subclones were compared for epitope specificity, T cell receptor variable gene expression and for various functional activities, in order to delineate properties crucial for pathogenicity. The Z1a-p and Znp cells expressed comparable levels of the T cell receptor genes and shared strong reactivity against the 72-84 epitope of MBP. The pathogenic Z1a-p cells displayed MBP-specific cytolytic activity in vitro, provided an in-vivo 'help' for elicitation of MBP-specific antibodies, mediated a delayed type hypersensitivity (DTH) response to MBP, caused EAE and vaccinated against the disease, thus demonstrating that a single CD4+ T cell clone is capable of eliciting various functions. The non-pathogenic Znp cells could also carry out most of these various functions, but failed to mediate a DTH response to MBP in normal animals. However, when inoculated into sublethally (650 R) irradiated syngeneic recipients, the Znp cells became highly pathogenic and mediated DTH response to MBP. Local irradiation of the recipient facilitated a DTH response to MBP in the irradiated ear, indicating that Znp cells are equipped with the effector mechanisms required for pathogenicity, and that their failure to cause disease may be accounted for by their inability to migrate into extravascular target tissue. Similar data were obtained with an independently isolated non-pathogenic clone, LB-3, specific for the encephalitogenic epitope of MBP. The ability of these non-pathogenic cells to vaccinate against EAE mediated by pathogenic cells raises the possibility that such non-pathogenic cells may play a role in triggering downregulation of pathogenic T cells.

Animals↗
PubMed1988

Evidence that TET protein functions as a multimer in the inner membrane of Escherichia coli.

The inner membrane TET (TetA) protein, which is involved in Tn10-mediated microbial tetracycline resistance, consists of two domains, alpha and beta, both of which are needed for tetracycline resistance and efflux (M.S. Curiale, L.M. McMurry, and S.B. Levy, J. Bacteriol. 157:211-217, 1984). Since tetracycline-sensitive mutants in one domain can partially complement sensitive mutants in the other domain and since some sensitive mutants show dominance over the wild type, a multimeric structure for TET in the membrane had been suggested. We have studied this possibility by using tetA-phoA gene fusions. We fused all but the last 40 base pairs of the tetA gene with the carboxy terminus of the phoA gene for alkaline phosphatase (PhoA), whose activity requires its dimerization in the periplasm. The tetA-phoA fusion protein was under control of the tetracycline-inducible regulatory system for the tetA gene. Induction led to the synthesis of a 78,000-dalton inner membrane protein. Tetracycline resistance was expressed at reduced levels, consistent with the terminal beta domain deletion. Alkaline phosphatase activity was also present, but at low levels, suggesting that some, but not all, of the fusion proteins had their carboxy-terminal ends in the periplasm. When wild-type or mutant TET proteins were present in the same cell with the fusion protein, the tetracycline resistance level was affected (raised or lowered); however, phosphatase activity was reduced only when TET proteins with intact or near-intact beta domains were present. These findings suggest that TET functions as a multimer and that intact beta domains, on TET molecules in the heterologous multimer, either allow fewer PhoA moieties to project into the periplasm or sterically hinder PhoA moieties from dimerizing.

Alkaline Phosphatase↗
PubMed1995

Identification, cloning, sequencing, and overexpression of the gene encoding proclavaminate amidino hydrolase and characterization of protein function in clavulanic acid biosynthesis.

Proclavaminate amidino hydrolase (PAH) catalyzes the reaction of guanidinoproclavaminic acid to proclavaminic acid and urea, a central step in the biosynthesis of the beta-lactamase inhibitor clavulanic acid. The gene encoding this enzyme (pah) was tentatively identified within the clavulanic acid biosynthetic cluster in Streptomyces clavuligerus by translation to a protein of the correct molecular mass (33 kDa) and appreciable sequence homology to agmatine ureohydrolase (M.B.W. Szumanski and S.M. Boyle, J. Bacteriol. 172:538-547, 1990) and several arginases, a correlation similarly recognized by Aidoo et al. (K. A. Aidoo, A. Wong, D. C. Alexander, R. A. R. Rittammer, and S. E. Jensen, Gene 147:41-46, 1994). Overexpression of the putative open reading frame as a 76-kDa fusion to the maltose-binding protein gave a protein having the catalytic activity sought. Cleavage of this protein with factor Xa gave PAH whose N terminus was slightly modified by the addition of four amino acids but exhibited unchanged substrate specificity and kinetic properties. Directly downstream of pah lies the gene encoding clavaminate synthase 2, an enzyme that carries out three distinct oxidative transformations in the in vivo formation of clavulanic acid. After the first of these oxidations, however, no further reaction was found to occur in vitro without the intervention of PAH. We have demonstrated that concurrent use of recombinant clavaminate synthase 2 and PAH results in the successful conversion of deoxyguanidinoproclavaminic acid to clavaminic acid, a four-step transformation. PAH has a divalent metal requirement, pH activity profile, and kinetic properties similar to those of other proteins of the broader arginase class.

ATP-Binding Cassette Transporters↗
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↗
PubMed1967

Studies with hydroxyurea. VII. Hydroxyurea and the synthesis of functional proteins.

Hydroxyurea affected neither the synthesis nor the degradation of bacterial messenger-ribonucleic acid. The proteins made by hydroxyurea-treated cells were structurally intact and fully functional. Since the expression of the lethal action of hydroxyurea requires active protein production, the data indicate that treated cells do not die as the result of the synthesis of abnormal proteins.

Alkaline Phosphatase↗
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↗
PubMed1986

ATP-coupled transport of vesicular stomatitis virus G protein. Functional boundaries of secretory compartments.

The oligosaccharide processing intermediates of the vesicular stomatitis virus strain ts045 G protein were used to identify ATP- and temperature-sensitive steps in the constitutive pathway of protein transfer to the cell surface. In addition to the initial ATP-sensitive step required for export from the endoplasmic reticulum (Balch, W. E., Elliott, M. M., and Keller, D. S. (1986) J. Biol. Chem. 261, 14681-14689), two distinct ATP-sensitive steps functionally dissect the Golgi into at least 3 compartments: a cis compartment containing the trimming enzyme mannosidase I, a medial compartment conferring resistance to endoglycosidase H, and a trans compartment containing terminal glycosyl transferases. A fourth ATP-sensitive step is required for export of G protein from the trans Golgi to the cell surface. A high threshold of cellular ATP (70% of the control) was required for maximal rates of transport between Golgi compartments. Transport between compartments is inhibited at 40% of the normal cellular ATP pool. Only a single temperature-sensitive step localized to the endoplasmic reticulum inhibited transport of ts045 G protein to the cell surface. The data suggest that ATP-sensitive steps punctuate transport of protein between compartmental boundaries of the secretory pathway.

Adenosine Triphosphate↗
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↗
PubMed1995

Abrogation of retinoblastoma protein function by c-Abl through tyrosine kinase-dependent and -independent mechanisms.

The decision to enter the cell division cycle is governed by the interplay between growth activators and growth inhibitors. The retinoblastoma protein (RB) is an example of a growth inhibitor whose main function appears to be the binding and inactivation of key cell cycle activators. One target of RB is a proto-oncoprotein, the c-Abl tyrosine kinase. RB binds to the ATP-binding lobe in the kinase domain and inhibits the nuclear pool of c-Abl in quiescent and G1 cells. Phosphorylation of RB at G1/S releases c-Abl, leading to the activation of this nuclear tyrosine kinase. In this report, we describe the construction of a mutant Abl, replacing the ATP-binding lobe of c-Abl with that of c-Src. The mutant protein AS2 is active as a tyrosine kinase and can phosphorylate Abl substrates, such as the C-terminal repeated domain of RNA polymerase II. AS2, however, does not bind to RB, and its activity is not inhibited by RB. As a result, the nuclear pool of AS2 is no longer cell cycle regulated. Excess AS2, but not its kinase-defective counterpart, can overcome RB-induced growth arrest in Saos-2 cells. Interestingly, wild-type c-Abl, in both its kinase-active and -inactive forms, can also overcome RB. Furthermore, overexpression of a kinase-defective c-Abl in rodent fibroblasts accelerates the transition from quiescence to S phase and cooperates with c-Myc to induce transformation. These effects, however, do not occur with the kinase-defective form of AS2. Thus, the growth-stimulating function of the kinase-defective c-Abl is dependent on the binding and the abrogation of RB function. That RB function can be abolished by the overproduction of one of its binding proteins is consistent with the hypothesis that RB induces cell cycle arrest by acting as a "molecular matchmaker" to assemble protein complexes. Exclusive engagement of RB by one of its many targets is incompatible with the biological function of this growth suppressor protein.

Adenosine Triphosphate↗
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↗
PubMed1982

Cellular folate binding proteins; function and significance.

It appears that specific functions may be assigned to some of the cellular folate binding proteins with some degree of certainty. Those that are membrane bound or derived from membranes probably have a role in transport of folate molecules into the cell. This is in spite of the fact that the localization of this protein to the plasma membrane has been carried out in only a limited number of cases. The role of the folate binding protein of L. casei in transport is much clearer. Bacteria provide the opportunity to obtain mutants defective in both transport and binding, and such mutants are more difficult to obtain with mammalian cell lines. The intracellular folate binding proteins have been discovered so far only in liver. The fact that the folate binding proteins in rat liver mitochondria are two enzymes, dimethylglycine dehydrogenase and sarcosine dehydrogenase, suggests that enzyme activities may eventually be discovered for the other intracellular folate binding proteins. This may not possible, however, and a reasonably strong case has been made that the folate binding protein of cytosol, FBP-CII, serves in a storage role. Such a storage role is difficult to prove since it depends, in part, on the demonstration that the protein becomes progressively less saturated during deficiency--a situation true also for enzymes.

Animals↗
PubMed1994

Perspectives on zinc finger protein function and evolution--an update.

Complexity is one of the hallmarks that applies to C2H2 type zinc finger proteins (ZFPs). Structurally distinct clusters of zinc finger modules define an extremely large superfamily of nucleic acid binding proteins with several hundred, perhaps thousands of different members in vertebrates. Recent discoveries have provided new insights into the biochemistry of RNA and DNA recognition, into ZFP evolution and genomic organization, and also into basic aspects of their biological function. However, as much as we have learned, other fundamental questions about ZFP function remain highly enigmatic. This essay is meant to define what we personally feel are important questions, rather than trying to provide a comprehensive, encyclopaedic review.

Amino Acid Sequence↗