Evidence for a KCl-Stimulated, Mg²⁺-ATPase on the Golgi of Corn Coleoptiles

Abstract

Membranes of corn (Zea may:, cv Trojan 929) coleoptiles were fractionated by sucrose density gradient ce action the locations of organelles were determined using marker enzymes and electron microscopy. Latent IDPase (or UDPase) was selected as the Golgi marker and UDPG-sterol glucosyl transferase ws selected as the plasma membrane (PM) marker, because they were clearly separable from markers for the other organelies. Golgi-rich and PM-rich fractions were studied in relation to their ATPase activities. The pH optimum of the KCI, MgeATPase of the PM-rich fraction from a step gradient was 6.0 to 65, while the Golgi-rich fraction had peaks at pH 6.0 to 6.5 and pH 7.5. It is hypothesized that the peak at pH 6.0 to 6.5 for the Golgi-rich fraction is due to PM-contamination, while the peak at pH 7.5 represents the activity of a Golgi ATPase. To The present work was undertaken in an effort to clarify the status of ATPases on Golgi membranes by careful comparison of ATPase activity and the distribution of various membrane markers on isopycnic and rate-zonal sucrose and dextran gradients. The corn coleoptile was selected because it is a well characterized system which allows reasonably good separation of membrane markers on gradients.Membrane ATPases have been implicated in the transport of ions in plant cells (34). On the basis of electrophysiological and biochemical evidence, the primary transport mechanism on the plasma membrane appears to be an electrogenic H+-ATPase which pumps protons to the cell exterior (41). There is also strong evidence that the tonoplast is the site of a second H4-ATPase which pumps protons into the vacuole (9,15,26,45).Ion transport in the Golgi has received very little attention, since its primary function is to secrete macromolecules across the plasma membrane. Inasmuch as many of these secreted macromolecules are charged, e.g. pectins and glycoproteins, charge balancing and pH adjustment may be required for normal processing of secreted macromolecules

The N-1-Naphthylphthalamic Acid-Binding Protein Is an Integral Membrane Protein

“…It is well established that the NPA-binding activity co-purifies with PM markers. Indeed, after the initial study by Lembi et al (1971), NPAbinding activity has often been used as a marker of PM fractions (Chanson et al, 1984;Hodges and Mills, 1988). In our material, the specific activity of the NPA-binding protein was enriched 3-fold in the PM fraction when compared to the total membrane fraction (Table I).…”

Cellular Localization of Protoporphyrinogen-Oxidizing Activities of Etiolated Barley (Hordeum vulgare L.) Leaves (Relationship to Mechanism of Action of Protoporphyrinogen Oxidase-Inhibiting Herbicides)

“…Molar extinction coefficients of (a) 6.22 m-' cm-' at 340 nm for NADH or NADPH and (b) 21.1 mM-' cm-' at 550 nm for Cyt c were used. IDPase was measured by the method of Chanson et al (1984), and the latent IDPase activity was determined as the difference of the activity in the presence and absence of 0.1% digitonin.…”

Calcium Transport in Tonoplast and Endoplasmic Reticulum Vesicles Isolated from Cultured Carrot Cells

“…These were centrifuged at 100,000g for 3 h. Microsomal Vesicles. Modified procedures of Sze (26) and Chanson et al (4) were used to isolate membrane vesicles from whole cell homogenates. All isolation steps were conducted at 4C.…”