RESUMEN
The plasma membrane (PM) H(+)-ATPase has been proposed to play important transport and regulatory roles in plant physiology, including its participation in auxin-induced acidification in coleoptile segments. This enzyme is encoded by a family of genes differing in tissue distribution, regulation, and expression level. A major expressed isoform of the maize PM H(+)-ATPase (MHA2) has been characterized. RNA gel blot analysis indicated that MHA2 is expressed in all maize organs, with highest levels being in the roots. In situ hybridization of sections from maize seedlings indicated enriched expression of MHA2 in stomatal guard cells, phloem cells, and root epidermal cells. MHA2 mRNA was induced threefold when nonvascular parts of the coleoptile segments were treated with auxin. This induction correlates with auxin-triggered proton extrusion by the same part of the segments. The PM H(+)-ATPase in the vascular bundies does not contribute significantly to auxin-induced acidification, is not regulated by auxin, and masks the auxin effect in extracts of whole coleoptile segments. We conclude that auxin-induced acidification in coleoptile segments most often occurs in the nonvascular tissue and is mediated, at least in part, by increased levels of MHA2.
Asunto(s)
Isoenzimas/metabolismo , ATPasas de Translocación de Protón/metabolismo , Zea mays/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Membrana Celular/enzimología , Clonación Molecular , ADN de Plantas/genética , Inducción Enzimática/efectos de los fármacos , Genes de Plantas , Ácidos Indolacéticos/farmacología , Isoenzimas/biosíntesis , Isoenzimas/genética , Datos de Secuencia Molecular , ATPasas de Translocación de Protón/biosíntesis , ATPasas de Translocación de Protón/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Distribución Tisular , Zea mays/genética , Zea mays/crecimiento & desarrolloRESUMEN
Plasma membrane vesicles derived from corn (Zea mays L.) roots retain a membrane-bound H+-ATPase that is able to form a H+ gradient across the vesicle membranes. The activity of this ATPase is enhanced 2- to 3-fold when Triton X-100 or lysophosphatidylcholine is added to the medium at a protein:detergent ratio of 2:1 (w/w). In the absence of detergent, the ATPase exhibits only one Km for ATP (0.1-0.2 mM), which is the same as for the pumping of H+. After the addition of either Triton X-100 or lysophosphatidylcholine, two Km's for ATP are detected, one in the range of 1 to 3 [mu]M and a second in the range of 0.1 to 0.2 mM. The Vmax of the second Km for ATP increases as the temperature of the assay medium is raised from 15[deg]C to 38[deg]C. The Arrhenius plot reveals a single break at 30[deg]C, both in the absence and in the presence of detergents. In the presence of Triton X-100 the H+-ATPase catalyzes the cleavage of glucose-6-phosphate when both hexokinase and ADP are included in the assay medium. There is no measurable cleavage when the apparent affinity for ATP of the H+-ATPase is not enhanced by Triton X-100 or when 1 mM glucose is included in the assay medium. These data indicate that when the high-affinity Km for ATP is unmasked with the use of detergent, the ATPase can use glucose-6-phosphate and hexokinase as an ATP-regenerating system.
RESUMEN
The effects of dimethyl sulfoxide (20% v/v) on the phosphorylation of Ca(2+)-ATPase of the sarcoplasmic reticulum by Pi vary depending on whether or not a Ca2+ gradient is formed across the vesicle membranes. In the absence of a Ca2+ gradient the solvent promotes a large increase in the affinity for Pi. This increase is no longer observed after the formation of a Ca2+ gradient. The enzyme affinity for Mg2+ is practically the same in the presence and absence of a gradient. Addition of dimethyl sulfoxide leads to an increase of the enzyme affinity for Mg2+ both in the presence and in the absence of a gradient.