PubMed Health⌕ Search

Biomedical subjects

A R Nebreda

Publications and source records attributed to A R Nebreda.

45 records · Page 3Linked to original sources
PubMed1991

Biochemical and biological comparison of HIV-1 NEF and ras gene products.

Human immunodeficiency virus type 1 (HIV-1) NEF protein has been reported to share certain biochemical and structural properties with known oncoproteins like src or rats. To determine whether this is a general property of NEF from various HIV isolates, three different NEF proteins were expressed in Escherichia coli using a thermoinducible expression system previously exploited to overproduce functionally active p21 ras proteins. ras and NEF proteins expressed in this manner were evaluated in parallel to compare their biochemical and biological properties. In contrast to ras, our NEF protein preparations had no detectable GTP binding but showed autophosphorylation activity when incubated in the presence of either GTP or ATP. This putative autokinase activity was higher in NEF proteins containing threonine at position 15 than in those carrying alanine at that position. Two different NEF genes also failed to induce oncogenic transformation of permanently transfected NIH 3T3 cells under conditions that led to oncogenic transformation using activated ras genes. Also, unlike ras, the NEF gene products failed to induce meiotic maturation when injected into fully grown Xenopus oocytes.

Adenosine Triphosphate↗
PubMed1989

Structural and functional properties of ras proteins.

The ras proteins belong to a family of related polypeptides that are present in all eukaryotic organisms from yeast to human. Their extraordinary evolutionary conservation suggests that they have essential cellular functions, although their exact role remains unknown. Mutations in specific amino acids and overexpression of normal proteins have been linked to altered proliferation and/or differentiation and, particularly, to neoplastic processes. Mature ras proteins are located on the inner side of the plasma membrane, and their biochemical properties include binding and exchange of guanine nucleotides and GTPase activity. The favored hypothesis for ras function is that these proteins exist in an equilibrium between an inactive conformation (p21.GDP) and an active conformation (p21. GTP) in which they are able to interact with their as yet unknown cellular target or targets. Similarities in cellular location, structure, and biochemistry with other known regulatory (G) proteins suggest that they play a role in transduction of signals from the cell surface. The elucidation of the crystal structure of normal and transforming ras proteins and the identification of cellular proteins that interact directly with them (GAP, CDC25) or suppress some of their biological effects (Krev-1) have opened new avenues in the search for their elusive cellular targets and in the elucidation of the functional role of ras gene products.

Amino Acid Sequence↗
PubMed1989

Preparation, characterization and properties of monoclonal antibodies against intact H-ras p21 proteins.

Monoclonal antibodies against intact normal (Gly12) and transforming (Lys12) BALB p21 ras proteins have been prepared and characterized. Their reactivity against the purified antigen proteins as well as against cellular ras proteins present in cell lines transformed by various alleles of ras genes was determined. Although some of the antibodies recognized preferentially either the normal or the activated form of BALB p21, none of them recognized exclusively either one. Deletion fragments of Ha-ras proteins were used in ELISA to determine the region of the protein encompassing the epitope recognized by each antibody. Two regions (amino acid stretches 23-69 and 89-106) accounted for the epitopes of about 75% of the mAbs raised in this study, suggesting that they encompass the most exposed and immunogenic domains in native ras p21 proteins. The affinity of these antibodies for p21 was higher than that of other ras mAbs described previously and allowed for (i) more quantitative immunoprecipitation of ras proteins, (ii) the use of selected mAbs in immunoaffinity purification of ras proteins and (iii) improved immunohistochemical detection of p21 in fixed tissues (Ward et al., accompanying manuscript). Finally, one of the mAbs described here appeared to recognize specifically a related antigenic molecule besides ras p21.

Animals↗
PubMed1988

Oligomeric structure of p21 ras proteins as determined by radiation inactivation.

Using radiation inactivation we determined that p21 ras proteins exhibit an oligomeric target size when assayed both structurally and functionally. Similar target sizes of p21 in ras-transformed cells and in purified preparations of the protein suggested that its structure is homo-oligomeric. p21 monomers were destroyed by radiation with the same target size as the GTP binding activity, indicating the occurrence of a tight association allowing energy transfer between the monomers. Irradiation in the presence of GTP, dithiothreitol, or EDTA did not change the target size. Normal (Gly12) and transforming (Lys12) forms of the protein exhibited similar target sizes. The homo-oligomeric structure suggests that p21 ras proteins do not conform to the structure of monomeric alpha subunits in classical G proteins (alpha beta gamma heterotrimers) and establishes similarities with other homo-oligomeric proteins (such as Escherichia coli CRP) which acquire the active conformation through subunit reorientation upon nucleotide binding.

Cell Line, Transformed↗
PubMed1987

Heterogeneous glycosylation of the EXG1 gene product accounts for the two extracellular exo-beta-glucanases of Saccharomyces cerevisiae.

Two exo-beta-glucanases of glycoprotein nature can be detected in culture supernatants of Saccharomyces cerevisiae cells. These exo-beta-glucanases show different Mr values and kinetic properties, although they are immunologically related. Their carbohydrate content and the electrophoretic mobility of both endoglycosidase H-treated exo-beta-glucanases suggest that they share the same protein fraction. Studies at genetic level relate the production of both extracellular exo-beta-glucanases with the expression of a single-copy gene in S. cerevisiae. Expression of this gene in another yeast, Schizosaccharomyces pombe, demonstrates that it codes for a protein with exo-beta-glucanase activity whose heterogeneous N-glycosylation accounts for both extracellular exo-beta-glucanases of S. cerevisiae.

Carbohydrates↗
PubMed1986

Cloning of genes related to exo-beta-glucanase production in Saccharomyces cerevisiae: characterization of an exo-beta-glucanase structural gene.

The EXG1 gene of Saccharomyces cerevisiae was cloned and identified by complementation of a mutant strain (exg1-2) with highly reduced extracellular exo-beta-1,3-glucanase (EXG) activity. Two recombinant plasmids containing an overlapping region of 5.2 kb were isolated from a genomic DNA library and characterized by restriction mapping. The coding region was located by subcloning the original DNA inserts in a 2.7-kb HindIII-XhoI fragment. Exg+ strains and Exg- mutants transformed with yeast multicopy plasmids containing this DNA fragment showed an EXG activity 5- to 20-fold higher than for the untransformed Exg+ wild-type (wt) strains. The overproduced EXG had the same enzymic activity on different substrates, and showed the same electrophoretic behaviour on polyacrylamide gels and identical properties upon filtration through Sephacryl S-200 as those of the main EXG from Exg+ wt strains. The EXG1 gene transformed Schizosaccharomyces pombe, yielding extracellular EXG activity which showed cross-reactivity with anti-S. cervisiae EXG antibodies. A fragment including only a part of the EXG1 region was subcloned into the integrating vector YIp5, and the resulting plasmid was used to transform an Exg+ strain. Genetic and Southern analysis of several stable Exg- transformants showed that the fragment integrated by homology with the EXG1 locus. The chromosomal DNA fragment into which the plasmid integrated has a restriction pattern identical to that of the fragment on which we had previously identified the putative EXG1 gene. Only one copy of the EXG1 gene per genome was found in several strains tested by Southern analysis. Furthermore, two additional recombinant plasmids sharing a yeast DNA fragment of about 4.1 kb, which partially complements the exg1-2 mutation but which shows no homology with the 2.7-kb fragment containing the EXG1 gene, were also identified in this study. This 4.1-kb DNA fragment does not appear to contain an extragenic suppressor and could be related in some way to EXG production in S. cerevisiae.

Chromosome Mapping↗
PubMed1983

Post-secretional modification of exo-1,3-beta-D-glucanases from Saccharomyces cerevisiae.

Exo-1,3-beta-D-glucanase secreted by Saccharomyces cerevisiae undergoes extracellular modifications in its carbohydrate moiety that change the affinity towards the lectin concanavalin A. The transition of negatively reacting enzyme form into positively reacting one depends on temperature. Results from experiments with glucono-delta-lactone and from treatments in vitro with hydrolases suggest a glycosidase-mediated mechanism.

Concanavalin A↗