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J E Allende

Publications and source records attributed to J E Allende.

At least 19 recordsLinked to original sources
PubMed1992

Folylpolyglutamate analogs can inhibit casein kinase II from Xenopus laevis.

Polyglutamate analogs of folate and related compounds were tested as inhibitors of casein kinase II (CK II) obtained from Xenopus laevis. The inhibitory capacity of the pteroyl, 4-amino-10-methyl pteroyl (the methotrexate aromatic moiety), and p-aminobenzoil derivatives increased as the number of gamma-glutamates attached went from 2 to 7. The nature of the aromatic head, group was also important since hexa-gamma-glutamatic acid had no inhibitory activity while the folylhexaglutamate derivatives were strong inhibitors with relative potency of methotrexate greater than pteroyl greater than p-aminobenzoic acid. The inhibition of CK II by methotrexate gamma-pentaglutamate was competitive with casein and showed an apparent K(i) of 90 microM.

Animals↗
PubMed1992

Differential stimulation of the GTPase activity of G-proteins by polylysine.

Polylysine, polyornithine and, to a lesser extent, polyarginine were found to stimulate the GTPase activity of the purified recombinant alpha subunit of the human G(i)-3 transducing protein alpha i-3. Optimal stimulation of 4- to 5-fold was obtained with polylysine concentrations between 1 and 20 microM, higher concentrations being inhibitory. Polylysine at similar concentrations stimulated by 50% the GTPase of transducin (GT), the vision transducing protein, but had only a very slight effect on the GTPase of the p21 product of the H-ras protooncogene. The stimulation of the alpha i-3 GTPase caused by polylysine was due to a reduction of the apparent Km for GTP from 3.8 to 1.3 microM. The stimulation by polylysine was observed at free Mg2+ concentrations below 1 microM. These results indicate that polylysine acts in a fashion similar to mastoparan and substance P in mimicking the action of an agonist-bound receptor on G-proteins.

Enzyme Activation↗
PubMed1992

The cDNAs coding for the alpha- and beta-subunits of Xenopus laevis casein kinase II.

Using a lambda gt10 cDNA library obtained from Xenopus laevis oocytes and probes derived from the known sequences of the human and Drosophila genes, a cDNA coding for the alpha-subunit of the X. laevis casein kinase II was isolated. The coding sequence of this clone determines a polypeptide of 350 amino acids. The X. laevis sequence is 98% identical to the human and rat proteins in the first 323 amino acids. Using the polymerase chain reaction to generate a 370-nucleotide-long probe, it was possible to clone and sequence a cDNA of 900 nucleotides that coded for the X. laevis beta-subunit of casein kinase II. The derived protein sequence is 215 amino acids long and again shows an extraordinary degree of conservation with other species.

Amino Acid Sequence↗
PubMed1991

Structure and function of G proteins.

G proteins are heterotrimeric proteins involved in the transduction of a variety of external signals in all eukaryotic organisms. This review analyzes the molecular aspects of G protein structure and function. The cloning of cDNAs coding for a great variety of G protein subunits has allowed us to deduce the primary and secondary structure of the subunits. Emphasis is given to the dissection of the molecular regions of the G alpha subunits implicated in the binding and hydrolysis of GTP and in the interaction with the receptor, with the effector and with the beta gamma dimer. The localization of these regions in a two-dimensional model of the G alpha subunit is attempted to provide a more comprehensive view of the structure and function of G proteins.

Amino Acid Sequence↗
PubMed1991

Polylysine activates membrane-bound adenylyl cyclase from Xenopus laevis oocytes through the Gs transducing protein.

1. The activity of the adenylyl cyclase found in the membranes of Xenopus laevis can be affected by polylysine and other polycations. 2. The activity of the catalytic subunit measured with forskolin is inhibited by polylysine and polyarginine at concentrations above 10 microM and by spermine above 3 mM. 3. The adenylyl cyclase activity stimulated by GTP-gamma-S or F- through the stimulatory G protein (Gs) can be increased by polylysine, polyornithine and spermine but not by polyarginine. 4. Polylysine stimulation of Gs dependent activity is due to the increase in the apparent affinity for GTP-gamma-S and to a lowering of the requirement for Mg2+ concentration.

Adenylyl Cyclases↗
PubMed1991

The hydrolysis of phosphatidylinositol 4-phosphate in membranes of Xenopus laevis oocytes: characteristics of a phosphomonoesterase.

1. Phosphatidylinositol 4-phosphate (PtdIns4P) is degraded by isolated membranes from Xenopus laevis oocytes. 2. Incubation of [4-32P]PtdIns4P with membranes yields only radioactive inorganic phosphate, indicating the presence of a phosphomonoesterase. 3. Membranes hydrolyze Ptd[2-3H]Ins4P to produce mainly Ptd[2-3H]Ins in the lipid phase. In this incubation [3H]inositol and inositol monophosphate appear in the water phase. 4. Membrane incubations of Ptd[2-3H]Ins4P carried out in the presence of excess non-radioactive Ins(1,4)P2 allows the trapping of small amounts of [3H]Ins(1,4)P2. These results demonstrate the presence of a phospholipase C. 5. Testing several phosphorylated analogs, it is determined that fructose 1,6-bisphosphate and alpha-glycerophosphate are potent inhibitors of the oocyte PtdIns4P phosphomonoesterase.

Animals↗
PubMed1990

Copolymers of glutamic acid and tyrosine are potent inhibitors of oocyte casein kinase II.

Polypeptides rich in glutamic acid are strong inhibitors purified from isolated nuclei of Xenopus laevis oocytes of casein kinase II. The presence of tyrosine in these peptides greatly enhances their inhibitory capacity. Using casein as a substrate, copolyglu:tyr (4:1) has an I50 value of 20 nM, 250 fold lower than that of polyglutamic acid which is 5 microM. A similar large difference is observed when a synthetic peptide is used as substrate. The inhibition of copolyglu:tyr is competitive with casein and can be completely reversed by high ionic strength. The relative inhibitory capacity of the polypeptides tested, in descending order, is copolyglu:tyr (4:1) greater than copolyglu:tyr (1:1) greater than polyglu greater than copolyglu:phe (4:1) greater than copolyglu:ala (6:4) greater than copolyglu:leu (4:1). The high affinity for tyrosine-containing acid peptides is shared by rat liver and yeast casein kinase II so that it seems to be a general property of these enzymes.

Animals↗
PubMed1990

The incorporation of myo-inositol into phosphatidylinositol derivatives is stimulated during hormone-induced meiotic maturation of amphibian oocytes.

The incorporation of myo-[3H]inositol into phosphatidylinositol and its phosphorylated derivatives was studied by microinjection of the radioactive precursor into Xenopus laevis oocytes. Induction of meiotic maturation of the oocytes by treatment with either progesterone one or insulin resulted in a significant increase in the incorporation of myo-[3H]inositol into the phospholipid fraction. This increase occurred 3-6 h after hormonal treatment, a time coincident with the start of the breakdown of the nuclear envelope, and requires protein synthesis. The effect of progesterone and insulin contrasts with the effect of acetylcholine, which acts through a muscarinic receptor causing the activation of phospholipase C, since the latter effector causes an increase in myo-[3H]inositol incorporation, which is more rapid and does not require protein synthesis. These results suggest that the meiotic maturation process is connected with changes in inositol metabolism in the amphibian oocyte.

Acetylcholine↗
PubMed1989

Nucleic acids can regulate the activity of casein kinase II.

Casein kinase II purified from nuclei of Xenopus laevis oocytes is inhibited by several specific nucleic acids. This kinase, the main phosphorylating activity of the oocyte nucleus, is markedly inhibited by poly U at 10 micrograms/ml, and this polymer is a competitive inhibitor of the phosphorylation of the substrate casein (Kiapp 80 nM). M 13 phage ssDNA and unfractionated yeast tRNA also inhibit between 50 and 200 micrograms/ml. Poly C, poly A, poly AG, dsDNA and Escherichia coli rRNA do not alter activity significantly at similar concentrations. Inhibitions are reversed by RNase (poly U, tRNA) or S1 nuclease (ssDNA). Oocyte casein kinase I or rabbit cAMP-dependent protein kinase are not inhibited by poly U at 200 micrograms/ml. The sensitivity of the casein kinase II to these inhibitors suggests a regulatory role for nucleic acids in nuclear phosphorylation reactions.

Animals↗
PubMed1989

Molecular cloning and sequence determination of a cDNA coding for the alpha-subunit of a Go-type protein of Xenopus laevis oocytes.

Xenopus laevis oocytes are cells ideally suited to the study of signal transduction and of the G-proteins that are involved in this process. A X. laevis cDNA library in lambda gt10 has been screened with a mixture of three oligonucleotide probes designed to detect sequences found in various mammalian alpha-subunits of G-proteins. One of these clones has been purified through tertiary screening and the DNA insert has been sequenced. This clone was found to include the total sequence coding for a 354 amino acid protein that is 89% identical to the sequence of alpha-subunit of rat Go. The differences with the mammalian protein were clustered in amino acids 290-315, which have been postulated to define the region interacting with the receptor and effector molecule. The homology with the alpha-subunits of other mammalian G-proteins is lower (65-70% to Gi and 42% to Gs). On this basis, this clone can be classified as Go-like.

Amino Acid Sequence↗
PubMed1989

Factors that regulate the activity of the phosphatidylinositol kinase present in oocyte membranes of Xenopus laevis.

1. Phosphatidylinositol kinase present in the membranes of Xenopus laevis oocytes was characterized. 2. The enzyme requires Mg2+ or Mn2+ at 10 mM and exogenous phosphatidylinositol (50 microM) increases the formation of phosphatidylinositol-4-phosphate. 3. The oocyte phosphatidylinositol kinase cannot use GTP as a phosphate donor but this compound inhibits competitively the utilization of ATP. 4. Addition of phosphatidylserine and phosphatidylinositol-4,5-bisphosphate stimulates the phosphorylation of phosphatidylinositol but 2,3-bisphosphoglycerate at 5 mM concentration is a strong inhibitor of the reaction.

1-Phosphatidylinositol 4-Kinase↗
PubMed1988

GTP-mediated macromolecular interactions: the common features of different systems.

G proteins that serve to transduce external signals in membranes share with protein synthesis factors and tubulin structural and functional features that are common to proteins that participate in reversible GTP-mediated macromolecular interactions. These proteins can bind GTP and GDP with high affinity, adopting different structures depending on whether they are complexed with the nucleotide diphosphate or triphosphate. The GTP.protein complex has high affinity for an acceptor macromolecule (or complex of macromolecules) and interacts with it, affecting its activity. These GTP-binding proteins also possess an intrinsic GTPase activity that is generally stimulated by its interaction with the acceptor. The GTPase activity converts the bound GTP to GDP, switching the configuration of the complexed protein to one of low affinity for the acceptor and causing its dissociation. The protein.GDP complex must exchange its GDP for GTP to allow the protein to acquire the high-affinity structure that can cycle back to the acceptor macromolecule. This exchange of guanine nucleotides requires in several instances exchange factors that can regulate the whole process. A detailed comparison of the features of the different systems is made with respect to structural similarities, regulation by protein phosphorylation, ADP ribosylation by bacterial toxins, and requirements for exchange factors. It is also proposed that there is a similar mechanism that involves ATP/ADP-binding proteins.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1988

Polylysine and polyamine stimulation of the phosphatidylinositol kinases of amphibian oocyte membranes.

Phosphatidylinositol kinase present in Xenopus laevis oocyte membranes catalyzes the formation of phosphatidylinositol 4-phosphate using phosphatidylinositol and ATP as substrates while the activity of a second enzyme, phosphatidylinositol-4-phosphate kinase, results in the synthesis of phosphatidylinositol 4,5-bisphosphate. Large (Mr greater than 20,000) homopolymers of L-lysine or L-ornithine can stimulate the activity of both of these enzymes by at least 2-fold at 10-20 microM concentrations. Under similar conditions poly-L-arginine fails to stimulate the reaction causing a partial inhibition. Smaller polylysine (25 lysines) or lysine-rich oligopeptides such as one corresponding to the last 14 amino acids of the carboxyl end of c-Ki-ras 2 protein produce appreciable stimulation of phosphatidylinositol but at concentrations of 300-500 microM. Spermine and spermidine at millimolar concentrations also stimulate exogenous phosphatidylinositol phosphorylation. The amino-glycoside antibiotic neomycin has a biphasic effect, stimulating the phosphatidylinositol kinase at concentrations below 0.5 mM and strongly inhibiting at higher concentrations. Polylysine also moderately stimulates the loss of radioactivity of phosphatidylinositol-4-[32P] phosphate observed in oocyte membranes. Polylysine and polyornithine do not change the apparent Km for ATP of the phosphatidylinositol kinase but increase the Vmax of the reaction.

1-Phosphatidylinositol 4-Kinase↗
PubMed1979

Studies on aminoacyl tRNA synthetases and transfer RNA in living Xenopus laevis oocytes.

The microinjection of transfer RNA into amphibian oocytes permits one to study under in vivo conditions the reactions that affect this important macromolecule. A comparative study has been carried out between the in vivo and in vitro specificity of the aminoacylation reacton. The results obtained show that modifications of the tRNA structure affect aminoacyl-tRNA synthetase recognition in the same fashion in both conditions. The in vivo aminoacylation was not affected by the presence of puromycin (0.5mM) or cycloheximide (0.1 mM) which completely inhibited oocyte protein synthesis. An interesting difference was obtained between the in vivo and in vitro aminoacylation of tRNA with regards to temperature requirements. While the in vivo reaction was optimal at 25 degrees and was totally inhibited at 37 degrees, the in vitro was optimal, at the latter temperature. The inhibition of the in vivo reaction at 37 degrees was not due to inactivation of the enzyme. The transfer of the amino acid moiety to nascent proteins was studied by measuring the transfer of radioactivity from injected (14C) phenylalanyl-tRNA into hot trichloroacetic acid precipitable material. It was found that 30% or more of the amino acid became incorporated into oocyte proteins and that this incorporation was due to direct transfer from the aminoacyl-tRNA and was inhibited by puromycin and cycloheximide.

Amino Acyl-tRNA Synthetases↗