Cytochemical localization of ATPase activity in oat roots localizes a plasma membrane-associated soluble phosphatase, not the proton pump

• Published in: Plant physiology (Bethesda) Vol. 86; no. 3; pp. 841 - 847
• Main Authors: Katz, D.B, Sussman, M.R, Mierzwa, R.J, Evert, R.F
• Format: Journal Article
• Language: English
• Published: Rockville, MD American Society of Plant Physiologists 01.03.1988
• Subjects: Adenosine triphosphatases; AVENA SATIVA; BIOCHEMISTRY; BIOCHIMIE; Biological and medical sciences; BIOQUIMICA; Cell biochemistry; Cell membranes; Cell physiology; CELL STRUCTURE; CYTOCHEMISTRY; Enzymes; ESTERASAS; ESTERASE; ESTERASES; ESTRUCTURA CELULAR; Fundamental and applied biological sciences. Psychology; HIDROLASAS; HYDROLASE; HYDROLASES; Lead; Molybdates; NUTRICION DE LAS PLANTAS; NUTRITION DES PLANTES; Oats; Phosphatases; Phosphates; PLANT NUTRITION; Plant physiology and development; Plants; Proton pumps; RACINE; RAICES; ROOTS; STRUCTURE CELLULAIRE; Avena sativa; Monocotyledones; Root; Enzyme; ATPase; Cytochemistry; Membrane pump; Enzymatic activity; Gramineae; Angiospermae; Plasma membrane; Spermatophyta; Localization; Proton transport
• ISSN: 0032-0889; 1532-2548
• DOI: 10.1104/pp.86.3.841

Cytochemical techniques employing lead-precipitation of enzymically released inorganic phosphate have been widely used in attempts to localize the plasma membrane proton pump (H+-ATPase) in electron micrographs. Using Avena sativa root tissue we have performed a side-by-side comparison of ATPase activity observed in electron micrographs with that observed in in vitro assays using ATPases found in the soluble and plasma membrane fractions of homogenates. Cytochemical analysis of oat roots, which had been fixed in glutaraldehyde in order to preserve subcellular structures, identifies an ATPase located at or near the plasma membrane. However, the substrate specificity and inhibitor sensitivity of the in situ localized ATPase appear identical to those of an in vitro ATPase activity found in the soluble fraction, and are completely unlike those of the plasma membrane proton pump. Further studies demonstrated that the plasma membrane H+-ATPase is particularly sensitive to inactivation by the fixatives glutaraldehyde and formaldehyde and by lead. In contrast, the predominant soluble ATPase activity in oat root homogenates is less sensitive to fixation and is completely insensitive to lead. Based on these results, we propose a set of criteria for evaluating whether a cytochemically localized ATPase activity is, in fact, due to the plasma membrane proton pump.