Malate Dehydrogenase Isoenzymes from Cotton Leaves

Abstract

Malate dehydrogenase isolated from leaves of the cotton plant (Gossypium hirsutum L.) appears in the form of several isoenzymes. Four of the isoenzymes found in cotton leaf ex- (24) in wheat, three (28) or four (21) in spinach, and in certain ornamental plants only one malate dehydrogenase enzyme has been detected (12). In cotton, at least three malate dehydrogenase isoenzymes have been reported (8, 27), but with crude extracts of cotton plants the exact number of isoenzymes could not be ascertained (27).In addition to this variation in the number of malate dehydrogenase isoenzymes which have been reported even from the same plant, a number of workers have found changes in malate dehydrogenase isoenzymes during plant development (5,12,14,15,27), or that the isoenzymes are localized in specific cell organelles (3,10,19,21,22,25,28), or that they have different molecular weights (5,11,15,25). Malate dehydrogenase isoenzymes have also been used as genetic markers with plants (4,19,22,24), apparently without regard for the difficulty of obtaining reproducible identification of the isoenzymes.One source of interest in the study of isoenzymes is the possible correlation of physiological function with individual isoenzymes. The belief that such a separation of functions may exist for plant malate dehydrogenase stems from the observation that malate exists in two pools in the plant cell (18). Unique metabolic roles in different parts of the plant cell have been suggested (21,28,29) as well as a role in salt uptake by plants (27).The considerable confusion regarding malate dehydrogenase isoenzymes outlined above and the interest which would attach to a better understanding of these isoenzymes prompted this study of the malate dehydrogenase isoenzymes of the cotton plant. Isoenzyme is here broadly defined to include all proteins with malate dehydrogenase activity. MATERIALS AND METHODSMalate dehydrogenase (E.C. 1 . 1 .1 . 37) isoenzymes were obtained from the leaves of cotton (Gossypium hirsutum L., var. Delta Pine 16). Plants were grown in a temperaturecontrolled glasshouse (29 C maximum, 21 C min.) under natural light. Leaves from one group of 6-to 8-week-old plants were generally used. Leaves were excised, and the petioles were placed in distilled water for transport to the laboratory. Extraction commenced within 20 min.Extraction and Ammonium Sulfate Precipitation. For extraction, petioles were removed, 10 g of leaf blades were weighed and counted. The leaves were rolled and cut into pieces approximately 0.5 X 0.5 cm with a scissors. All succeeding operations were performed in the cold at 0 to 4 C.The chopped leaves were placed in an 80-ml container of a Lourdes homogenizer and sprinkled with 5.0 g of Polyclar AT. To this was added 40 ml of extraction buffer consisting of 50 mm tris-HCl, 10 mm mercapthoethanol and 1 mM EDTA at pH 7.5. Homogenization consisted of three periods of 10 sec at 60 v with 20-sec intervals. The ground slurry was squeezed through a nylon bag with a 25 X 35 threads/cm2 mesh and an addi...

Purification and molecular properties of malate dehydrogenase from the marine diatom Nitzschia alba

“…On the other hand, the sequence further downstream (residues 22-32) shows a high degree of diversity among pig heart mitochondrial, E. coli and N. alba MDH enzymes. It has been shown that this particular region (residues [22][23][24][25][26][27][28][29][30][31][32] contains no significant functional domain [50]. …”

“…In crude extracts it was shown that MDH might exist as oligomeric forms with the Mr much higher than the usual 60000-70000, as indicated by non-denaturing polyacrylamide/starch-gel electrophoresis and gel-filtration column chromatography [21]. These high-Mr MDH species, usually in addition to the normal-Mr form of the enzyme, have been reported in bacteria [1,2], fungi [6], animal mitochondria [22] and, most commonly, in higher plants [21,23,24]. These results indicated that the higherMr species of MDH in higher plants might result from aggregation of low-Mr species.…”

Malate Dehydrogenase Isoenzymes in Division Synchronized Cultures of Euglena

“…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).…”

Glyoxysomal malate dehydrogenase and malate synthase from soybean cotyledons (Glycine max L.): enzyme association, antibody production and cDNA cloning

“…It now appears that previous studies on the aggregation behavior of MS under low-ionic-strength conditions overlooked its possible association with gMDH and that the decameric MS structure postulated by Henry et al (1992) is likely to correspond to an association between octameric MS and tetrameric gMDH. Our finding of MDH activity eluting as complexes of high Mr (670 kDa and 140 kDa) is not unusual; indeed, elution profiles of plant MDHs with apparent high-Mr values of 500kDa and 280kDa have been reported (O'Sullivan and Wedding 1972;Habig and Racusen 1974). Although the high-Mr MDH isoforms are very similar to the most commonly found low-Mr isoform with respect to optimal pH, K m for malate, and inhibition by various unreactive substrate analogs, they differ in their electrophoretic properties and ability to reduce 3-acetylpyridine-deamino-NAD + (Habig and Racusen 1974).…”