Enzymes of the Moss Funaria hygrometrica II: The Isoenzymes of Malate Dehydrogenase

Abstract

Ten isoenzymes of malate dehydrogenase were present in wholecell extracts of the protonemal stage of the moss Funaria hygrometrica Hedw. They were isolated via disc gel electrophoresis and column chromatography. These isoenzymes could be separated only partially via fractionation of the cells into particulate and supernatant fractions. Three isoenzymes of malate dehydrogenase were found to utilize NADP as the cofactor. Malate dehydrogenase has a wide-ranging role in vascular plant metabolism. It is involved in the citric acid cycle (Harley & Beevers, 1963), heterotrophic carbon dioxide fixation (Lips & Beevers, 1966), peroxisomal activity (Yamazaki & Tolbert, 1969), and various types of photosynthesis (Rocha et al., 1968; Mukerji & Ting, 1969; Hatch et al., 1969). Various workers (Rocha et al., 1968; Yamazaki & Tolbert, 1969) have reported unique MDH isoenzymes for chloroplasts, mitochondria, peroxisomes, and the cytoplasmic fluid, with the total number of reported isoenzymes ranging from three to twelve (O'Sullivan & Wedding, 1972). We have therefore undertaken a study of this enzyme in the moss Funaria hygrometrica Hedw. A knowledge of the number and location of MDH isoenzymes should give us some insight into 1) what type of metabolic activities might be present in Funaria, and 2) the phylogenetic relationship of mosses to higher plants. MATERIALS AND METHODS Protonemal Growth.-Funaria hygrometrica sporophytes were collected locally. The spores were seeded by breaking the spore capsule over an agar plate. At times the capsules were first sterilized with 1% hypochlorite solution. Bristol's medium as modified by Bold (1949), adjusted to pH 7.8 for maximal protonemal growth (Armentano & Caponetti, 1972), was used throughout. The plates were incubated for about 10 days at 200C in continous light. The protonemata were then transferred to 350 ml liquid medium, and incubated with aeration for one week. The entire culture was then transferred to 3 liters of liquid medium and incubated another week with aeration. The yield was generally between 10-15 g wet weight. Extraction Procedure.-The protonemata were ground with mortar and pestle in acetone at 00C and the acetone powder collected over filter paper. This was stored in the freezer for up to one week. The powder was then suspended in borate buffer, pH 8.9 (0.1 M boric acid, 0.075 M sodium borate, 0.025 M sodium chloride) and centrifuged for 5 min. at 12,100 X g. The supernatant was collected via filtration and brought to 90% saturation with ammonium sulfate. The precipitate was collected by centrifugation at 14,500 x g for 20 min. 1 Supported by Brown-Hazen Fund of the Research Corporation, and the National Science Foundation, Undergraduate Research Participation Grant GY-5162. Research conducted at Dickinson College, Carlisle, Pennsylvania 17013. 2 Department of Biology, Eastern College, St. Davids, Pennsylvania 19087. This content downloaded from 157.55.39.45 on Fri, 02 Sep 2016 06:22:11 UTC All use subject to http://about.jstor.org/terms 578 THE BRYOLOGIST [Volume 77 Band Number Rm Value