Characterization of an ATPase Associated with the Inner Envelope Membrane of Amyloplasts from Suspension-Cultured Cells of Sycamore (Acer pseudoplatanus L.)

Abstract

Amyloplast envelope membranes isolated from cultured, white-wild cells of sycamore (Acer pseudoplatanus L.) have been found to contain a Mg2 -ATPase, ranging in specific activity from 5 to 30 nanomoles per minute per milligram protein. This ATPase hydrolyzes a broad range of nucleoside triphosphates, whereas it hydrolyzes nucleoside mono-and diphosphates poorly, if at all. The ATPase activity was stimulated by several divalent cations, including Mg2 , Mn2 and Ca2+, whereas it was not affected by Sr2 , K+, or Na . The Km for total ATP was 0.6 millimolar, and the activity showed a broad pH optimum between 7.5 and 8.0. The ATPase was insensitive to N,N'-dicyclohexylcarbodiimide and oligomycin, but it was inhibited by vanadate. All these characteristics are basically similar to those reported previously for the Mg2+-ATPase of the chloroplast inner-envelope membrane. Likewise, the amyloplast envelope enzyme was shown to be located specifically on the inner envelope membrane. The amyloplast envelope membranes were chemically modifled with a series of unique affinity labeling reagents, the adenosine polyphosphopyridoxals (M Tagaya, T Fukui 1986 Biochemistry 25: 2958-2964. About 90% of the ATPase activity was lost when the envelope membranes were preincubated with 0.1 millimolar adenosine triphosphopyridoxal. Notably, the enzyme was protected completely from inactivation in the presence of its substrate, ATP. In contrast, both adenosine diphosphopyridoxal and pyridoxal phosphate caused much less of an inhibitory effect. This greater relative reactivity of the triphosphopyridoxal analog is similar to that reported previously with Escherichia coli F1 ATPase (T Noumi et al. 1987 J Biol Chem 262: 7686-7692).The occurrence of a DCCD3-insensitive Mg2+-ATPase has been reported in the chloroplast envelope membranes from spinach (3,11,15,22,23), pea (5, 16-18, 21, 28) (8) reported the presence of a DCCDinsensitive Mg2+-ATPase apparently associated with the amyloplast envelope from etiolated epicotyls of Pisum sativum, the putative ATPase of the amyloplast envelope has not been well characterized. The aim of our study was to characterize the ATPase ofthe amyloplast envelope for the further understanding of its possible function(s) in this unique plastid-type. In addition, we have established a method for resolving the inner and outer membranes of the amyloplast envelope in order to determine the location ofthe ATPase, and we have found that it is localized on the inner membrane, similar to that in green-leafchloroplasts (3,17,18,21).Fukui, Tagaya, and coworkers (24,30,31) previously synthesized nucleoside polyphosphopyridoxal compounds and tested their potential affinity for several ATP-binding enzymes such as adenylate kinase and lactate dehydrogenase. The analysis of the modified enzymes indicated that these afflnity labels were exclusively bound to the active site lysyl residue of the enzyme with much higher specificity than that of pyridoxal phosphate. In this study, we also examined and compared PLP, AP2-PL, and AP...

Immunochemical Analysis Shows That an ATP/ADP-Translocator Is Associated with the Inner-Envelope Membranes of Amyloplasts from Acer pseudoplatanus L.

“…The profiles visualized by silver staining are quite different between them (lanes 3 and 6), and densitometric tracing clearly indicates that there are specific bands characteristic of each inner-membrane fraction (data not shown). It should be noted that, consistent with our previous reports (8,9,16), there is a major polypeptide (32-kD) (closed circle) in the amyloplast inner-membrane which is clearly separable from a faint band of 31-kD band corresponding to the Pitranslocator (see below and Fig. 3) (star), whereas the 3 l-kD protein is a dominant molecular species in the inner-membrane of the chloroplasts.…”

“…The method for separating the outer-and inner-envelope membranes from isolated intact amyloplasts and chloroplasts by the protocol originally developed by Cline et al (3) has been reported previously (8,9). The specific localization of Mg2+-ATPase in the inner and acyl-CoA synthetase in the outer-envelope membranes of plastids has been described in the previous paper (8,9).…”

“…The specific localization of Mg2+-ATPase in the inner and acyl-CoA synthetase in the outer-envelope membranes of plastids has been described in the previous paper (8,9). Total mitochondrial membranes were isolated by homogenizing intact mitochondria from white-wild cells in a hypotonic solution containing 10 mM Mops-NaOH (pH 7.8), 1 mM MgCl2, and 1 mM EDTA with a ground-glass homogenizer.…”

Characterization and Intraorganellar Distribution of Protein Kinases in Amyloplasts Isolated from Cultured Cells of Sycamore (Acer pseudoplatanus L.)

“…Isolation of suborganellar fractions from hypotonically lysed plastids was performed as described previously (12,13,19,21,22). The purity of the isolated fractions was assessed by comparison with the polypeptide patterns obtained after SDS-PAGE, which was reported previously (12,19,22), and by measuring the activity of the marker enzymes 6-PG4 dehydrogenase (stroma) and Mg2+-ATPase (envelope) (see below).…”

“…(3,6,7,(24)(25)(26). Among plant organelles, chloroplast polypeptides located in the thylakoids, stroma, and envelope have been shown to undergo reversible phosphorylation (3,6,7,9,12,14,17,24,27,28).…”