Malate Dehydrogenase Isoenzymes in Division Synchronized Cultures of Euglena

Abstract

(26)(27)(28). In addition, recent work has demonstrated a further isoenzyme of malate dehydrogenase localized in plant microbodies (22,23,29). Although the isoenzymes of malate dehydrogenase in higher plant tissues have been the subject of many investigations (11,(21)(22)(23)(28)(29)(30), little is known about the distribution and function of these isoenzymes in the algal cell. When Euglena cell organelles were separated by sucrose density gradient centrifugation, malate dehydrogenase activity was recorded in several fractions (17). Besides malate dehydrogenase activity in the mitochondrial fraction, activity was present in the particulate glycolate oxidoreductase fraction, the supernatant, but not in the chloroplast fraction (17). When grown phototrophically on an appropriate light-dark regime, cultures of Euglena gracilis divide synchronously, an appropriate doubling of cell number occurring in each dark period within certain limits of cell concentration (7). By using division synchronized cultures, the development of malate dehydrogenase activity in relation to other cellular activities can be investigated. In the present paper, we report on the characterization of malate dehydrogenase isoenzymes in cell fractions and their expression over the division cycle in Euglena cultures. MATERIALS AND METHODSGrowth, Synchronization Regime, and Sampling of Culture. Division synchronized cultures of E. gracilis Klebs strain Z were obtained exactly as described previously (4). As before, samples were removed at ts, t12, t17, and ti,, this referring to the hour of sampling after commencement of the 24-hr cycle in which the culture was used. Asynchronous cultures were grown at 25 C in the photoautotrophic growth medium of Cramer and Myers (9) at a continuous light intensity of 6000 lux provided by banks of fluorescent tubes (Osram white) and gassed with air at a rate of 71/hr. Cells were harvested in middle exponential phase of growth by centrifugation at 10OOg for 4 min.CeUl

The localization of glycollate-pathway enzymes in Euglena

“…After centrifuging at 250g for 5min to remove any remaining cell debris, the crude extract was used for the isolation or organelles. The method of disruption gave a marginally better yield of organelles than in previous experiments, when cells were shaken with ballotini in a Braun MSK rotary cell homogenizer (Davis & Merrett, 1973). All sucrose solutions were prepared in 20mM-glycylglycine buffer, pH 7.5.…”

Proteome of the secondary plastid of Euglena gracilis reveals metabolic quirks and colourful history

“…The plastid fraction was isolated as described previously (Davis and Merrett 1973;Moreno-Sánchez et al 2000;Dobáková et al 2015). Cells of E. gracilis strain SAG 1224-5/15 were collected by centrifugation at 800 × g for 10 min and resuspended in SHE buffer (250 mM sucrose, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1 mM ethylenediaminetetraacetic acid (EDTA), pH 7.3) supplemented with 0.4% fatty acid-free bovine serum.…”

“…The gradient was prepared by layering decreasingly dense sucrose solutions upon one another from 2.0, 1.75, 1.5, 1.25, 1, to 0.5 M, 5 ml each and was centrifuged in the SW-28 rotor at 87,041 × g (22,000 rpm) at 4 °C for 4.5 h (L8-M Ultracentrifuge, Beckman). Ultracentrifugation separated the plastids from mitochondria and peroxisomes (Davis and Merrett 1973). The plastid fraction was situated at the interface of the 1.5 and 1.25 M sucrose and was collected by a syringe.…”

Organelle isolation and partial characterization from giant cells ofAspergillus niger

“…Fumarase ([-C 4.2.1.2) was assayed spectrophotometrically by the method of Massey [8], malate dehydrogenase (EC 1.1.1.37) by the method of Davis and Merrett [9]. cytochrome c reductase (1{(" 1.…”