Tissue Distribution of Microbody, Mitochondrial, and Soluble Malate Dehydrogenase Isoenzymes

Many tissues which have been investigated have at least two malate dehydrogenase isoenzymes. One is an organelle form localized in mitochondrial fractions whereas the other does not appear to associate with organelles sedimenting after high speed centrifugation (17). Spinach leaf tissue has an additional malate dehydrogenase isoenzyme localized in the peroxisome or leaf microbody fraction (12, 19). Microbodies are single membrane, dense protein-containing bodies which occur in almost all plant tissues (3, 7). The leaf microbodies or peroxisomes are believed to function in photorespiration (16). It was important, therefore, to investigate microbody fractions isolated from nongreen spinach tissue (with no photorespiration) in order to ascertain if the peroxisomal or microbody malate dehydrogenase of spinach is exclusively a form associated with green tissue metabolism or if it occurs in microbodies of nongreen tissues as well. It should be noted that glyoxysomes, microbodies with the glyoxylate cycle metabolism, have malate dehydrogenase activity (2). Green stem tissue of Opuntia also has three distinctly different isoenzymes of malate dehydrogenase (10). Spinach seeds (Spinacia oleracea L. var Bloomsdale) were germinated in the dark at 25 C on moistened filter paper. From the dark-grown tissue, nongreen root and hypocotyl tissue and etiolated cotyledon tissue were obtained. In some experiments, the young seedlings were exposed to light in the greenhouse in order to obtain green cotyledon tissue. Mature green leaf tissue was either grown in the greenhouse or purchased locally. Tissue homogenates were obtained by grinding the tissue in 0.05 M tris buffer, pH 7.5, and precipitating the protein with solid ammonium sulfate (0-0.8 of saturation). The precipitate was resuspended by dialysis in 5 mm sodium phosphate buffer, pH 7.0. The resulting preparation was assayed for malate dehydrogenase by starch gel electrophoresis (5) or layered on analytical 1 x 10 DEAE2-cellulose anion exchange columns (1.5 x 15 cm of 0.61 meq/g) equilibrated with 5 mi sodium phosphate buffer (10, 11). Protein was eluted with a 0.02 to 0.2 M sodium phosphate gradient (14). Three-milliliter fractions were collected and assayed for malate dehydrogenase. In order to obtain intact organelle preparations, the various tissues were gently minced with a chopper in 0.5 M sucrose-0.05 M tris buffer (pH 7.5) containing 1 mm EDTA, 1 mm 2-mercaptoethanol, and 0.1 % bovine serum albumin. After mincing, the suspension was gently ground in a mortar for 30 sec. The resulting homgenate was filtered through eight layers of cheesecloth, and the filtrate was centrifuged at 250g for 90 sec to remove cellular debris. The supernatant containing intact organelles