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R Serrano

Publications and source records attributed to R Serrano.

At least 145 records · Page 8Linked to original sources
PubMed1987

Inhibition of the proton pumping ATPases of yeast and oat root plasma membranes by dicyclohexylcarbodiimide.

The inhibition of the proton-pumping ATPases of yeast and oat root plasma membranes by dicyclohexylcarbodiimide (DCCD) can be correlated with the covalent incorporation of the inhibitor. Full inhibition of the yeast enzyme required the incorporation of about 1 mol DCCD/mol of the ATPase polypeptide of 100 kDa. A kinetic study of the interaction of DCCD with the yeast and oat ATPases indicates a second-order rate constant of about 500 M-1 min-1 and a stoichiometry of 1 mol DCCD/mol of enzyme, in agreement with the amount of DCCD incorporated by the yeast enzyme. It is proposed that DCCD reacts with a single carboxylic group present in a hydrophobic region of these proton-pumping ATPases and which could participate in proton binding and transport.

Amino Acid Sequence↗
PubMed1987

Replacement of the promoter of the yeast plasma membrane ATPase gene by a galactose-dependent promoter and its physiological consequences.

In order to probe the physiological role of the yeast plasma membrane ATPase we have replaced the constitutive promoter of its gene by a galactose-dependent promoter. The resulting cells stop growing on glucose medium when the preformed ATPase is diluted to 20% of normal. There is a correlation between ATPase activity and both proton efflux from the cells and amino acid transport. A large proportion of growth-arrested cells appear enlarged and with several buds containing nuclei.

Adenosine Triphosphatases↗
PubMed1984

Purification of the proton pumping ATPase from plant plasma membranes.

The plasma membrane ATPase from oat roots has been purified near homogeneity by a simple procedure. Plasma membranes isolated from sucrose gradients are first extracted with Triton X-100 and KC1 and the residue solubilized with lysolecithin. Rate-zonal centrifugation in a vertical rotor with a glycerol gradient results in a preparation of very high specific activity (6 mumoles min-1 mg protein-1 at 30 degrees C) and where over 70% of the protein corresponds to a polypeptide of about 100 kilodaltons previously identified as the ATPase. The purified enzyme could be reconstituted in proteoliposomes catalyzing ATP-driven proton transport sensitive to vanadate.

Catalysis↗
PubMed1983

In vivo glucose activation of the yeast plasma membrane ATPase.

The addition of glucose to yeast cells activates proton efflux mediated by the plasma membrane ATPase. Accordingly, the ATPase activity of purified plasma membranes is increased up to 10-fold. The activated ATPase has a more alkaline pH optimum, better affinity for ATP and greater sensitivity to vanadate than the non-activated enzyme. All these changes are reversed by washing the cells free of glucose. This suggests two states of the ATPase which are interconverted by a covalent modification. As glucose does not affect the phosphorylation of plasma membrane polypeptides, other type of covalent modification may be involved.

Adenosine Triphosphatases↗
PubMed1983

Phosphorylated intermediate of the ATPase of plant plasma membranes.

A partially purified preparation of the plant plasma membrane ATPase was phosphorylated when incubated with [gamma-32P]ATP. The phosphoprotein formed has the characteristics of an enzyme intermediate because of its rapidity of phosphorylation and dephosphorylation. The sensitivity of the phosphoenzyme bond to alkaline pH and to hydroxylamine indicates that it is an acylphosphate. Both the ATPase activity and the phosphorylation of the enzyme exhibited an apparent Km value of 0.3 mM ATP. When the phosphorylated enzyme was analyzed by electrophoresis in sodium dodecyl sulfate, only one major band with a molecular weight of about 105,000 contained radioactivity. These results indicate that the plant plasma membrane ATPase has a subunit composition and reaction mechanism similar to the cation-pumping ATPases of animal and fungal plasma membranes.

Adenosine Triphosphatases↗
PubMed1983

Solubilization of yeast plasma membranes and mitochondria by different types of non-denaturing detergents.

A comparative study of the solubilization of yeast plasma membranes and mitochondria by different types of non-denaturing detergents has been performed. Zwittergent-14 (3-[tetradecyldimethylammonio]-1-propanesulfonate) at low concentrations (3-4 mM) produced maximum solubilization of both membranes. However, this detergent may inactivate enzymes at high concentrations. Taurodeoxycholate (in the presence of salt) and Triton X-100 were also effective in mitochondria but not in the plasma membranes. Octylglucoside only solubilized these membranes at very high concentrations (20 mM). CHAPS (3-[cholamidopropyldimethylammonio]-1-propanesulfonate) only achieved partial solubilization even at high concentrations. Our results suggest that Zwittergent-14 at low concentrations is one of the most powerful detergents for the general solubilization of native membrane proteins.

Cell Fractionation↗
PubMed1982

Partial purification and properties of the proton-translocating ATPase of plant plasma membranes.

The plasma membrane ATPase of plant cells has been postulated to operate as an electrogenic proton pump which derives the co-transport of nutrients with protons and which possibly catalyzes K+ transport (Poole, R. J. (1978) Annu. Rev. Plant Physiol. 29, 437-460). In addition, the enzyme seems to determine cell growth after hormonal stimulation by acidifying the external medium (Marré, E. (1979) Annu. Rev. Plant Physiol. 30, 273-288). In order to substantiate this important physiological role, the ATPase from oat root plasma membranes has been solubilized with a zwitterionic detergent and partially purified. A polypeptide of 93,000 daltons was enriched in the course of the purification. The enzyme was completely specific for ATP as substrate and it was inhibited by vanadate, diethylstilbestrol, and dicyclohexylcarbodiimide but not by oligomycin or ouabain. The ATPase activity was stimulated by K+ but this occurred only at acidic pH and the effect was less than 100%. After reconstitution of proteoliposomes by a freeze-thaw-sonication procedure, proton transport driven by ATP was demonstrated by the quenching of acridine dye fluorescence. Proton transport occurred in the absence of K+ and its electrogenic nature was demonstrated by the requirement for permeant ions (nitrate or K+ with valinomycin). It is suggested that the enzyme is an electrogenic proton pump, somewhat stimulated by K+, but not involved in the transport of this cation.

Adenosine Triphosphatases↗
PubMed1981

Purification and characterization of a membrane-bound ATP diphosphohydrolase from Cicer arietinum (chick-pea)roots.

Microsomal membranes from Cicer arietinum (chick-pea) roots contained an ATP phosphohydrolase activity that could be solubilized by high-ionic-strength media. The enzyme has been purified to homogeneity by affinity and ion-exchange chromatography. It has the properties of an ATP diphosphohydrolase (apyrase, EC 3.6.1.5) that hydrolyses different nucleoside di- and tri-phosphates but has no activity towards monophosphoric esters and pyrophosphate. No stimulation by K+ could be demonstrated for either the membrane-bound or the purified enzyme, and therefore it would seem not to be related to the K+ -dependent ATPase postulated to mediate K+ transport in plants.

Apyrase↗
PubMed1981

Phosphorylated intermediate of the ATPase from the plasma membrane of yeast.

A purified preparation of the plasma-membrane ATPase from Saccharomyces cerevisiae was phosphorylated when incubated with [gamma-32P]ATP. The phosphoprotein formed has the characteristics of an enzyme intermediate because of its rapidity of phosphorylation and dephosphorylation. When the phosphorylated enzyme was analyzed by polyacrylamide gel electrophoresis in sodium dodecylsulfate only one band with a molecular weight of 100000 contained radioactivity. This band represented about 80% of the protein of the preparation and its enrichment in the course of the purification correlated with the increase in the specific ATPase activity. Both the ATPase reaction and the phosphorylation of the enzyme exhibited an apparent dissociation constant for the enzyme-ATP complex of 0.2 mM, further implicating the phosphoenzyme as an intermediate of the reaction. The sensitivity of the phosphoenzyme bond to alkaline pH and hydroxylamine indicate that it is an acylphosphate. From the maximum level of intermediate (0.7 nmol/mg) and the maximum ATPase activity at 30 degrees C (21 mumol x min-1 x mg-1) a turnover number of 30000 min-1 can be calculated. The level of phosphoenzyme was not affected by either the ATPase inhibitors vanadate and dicyclohexylcarbodiimide or by ADP. These results indicate that the yeast plasma-membrane ATPase has a subunit composition and reaction mechanism similar to the cation-pumping ATPases of animal plasma membranes.

Adenosine Triphosphatases↗
PubMed1981

Reconstitution of the proton-translocating adenosine triphosphatase of yeast plasma membranes.

The plasma membrane ATPases of eukaryotic cells of the vegetable type (fungi, plants, and algae) have been postulated to operate as proton pumps which generate membrane potentials and drive the uptake of nutrients by proton co-transport (Poole, R. J. (1978) Annu. Rev. Plant Physiol. 29, 437-460). In order to verify this important physiological role, a purified preparation of the yeast plasma membrane ATPase has been reconstituted with soybean phospholipids by a freeze-thaw-sonication procedure. The reconstituted proteoliposomes catalyzed a 32Pi-ATP exchange partially sensitive to proton ionophores (uncouplers) and to the proton-potassium exchange carrier nigericin. The reaction was completely inhibited by the nonspecific ionophore gramicidin and by the combination of uncouplers with the potassium ionophore valinomycin. These results are interpreted as evidence for two types of proton transport catalyzed by the enzyme preparation: electrogenic proton transport and electroneutral proton-potassium exchange.

Adenosine Triphosphatases↗