A Site With an Affinity for Heavy Metals on the Thylakoid Membranes of Chloroplasts

Abstract

In experiments designed to localize adenosine triphosphatase activity on cellular membranes of the pea tendril we have localized a non-enzymic, discrete deposition of lead, osmium and tungsten at specific sites on the thylakoid membranes of chloroplasts. These reactive sites may be exposed to the stain or partially blocked depending on the nature of the aldehyde employed for preliminary fixation of the tissue. We have noted several published electron micro-graphs of chloropiast fine structure in which similar dense staining at these sites is evident. With few exceptions, the authors have ignored these deposits and their possible significance, undoubtedly owing to their very sporadic appearance in glutaraldehyde-fixed tissue. In pea tendrils fixed with hvdroxy-adipalde'nyde, such deposits appear consistently in the chloroplasts and the degree of staining with heavy metals at these sites is appreciably magnified. The present report describes some characteristics of this reaction. Materials and Methods Segments of tissue from the unibranched tendril arising at the fifth internode of light grown pea plants (Pisum sativum L. var. Alaska) were used for all experiments. The material was fixed in a solution of either 3 % (v/v) glutaraldehyde or 3 % (v/v) hvdroxvadipaldehyde in 0.07s5 m cacodylate buffer (pH 7.2) containing 8 % (w/v) sucrose and depending upon the experiment, with or without 0.001 M MgSO4 and 0.01 % CaCl1. Fixation took place at 2 to 40 for 30 min with glutaraldehyde or 45 min witlh hydroxyadipaldehyde. Both aldehydes were purified before use by filtration through layers of calcium carbonate and Norit A activated charcoal to remove acid and polymer impurities. Following fixation, the tissue was washed overnight (ca. 18 hr) in 2 changes of cacodvlate buffer and then incubated for 2 hr at pH 7.2 in the Waclhstein-Meisel (WM) medium (8) containing 1 mM lead nitrate. After incubation, the tissue was rinsed briefly in the caco-dvlate buffer and post-fixed in 1 % (w/v) osmic acid for 60 to 90 min at 2 to 40. Dehydration was effected with acetone and the material was embedded in Epon 812. Thin sections were cut with a diamond knife on a Sorvall MT-2 ultramicrotome and stained briefly (4 min) with Reynold's lead citrate. The grids were examined in a JEM 6C electron microscope operating at an accelerating voltage of 50 kv. Any deviations from this basic procedure are mentioned where appropriate in the text.