Abstract. Leaves of 10 plant species, 7 with photorespiration (spinach, sunflower, tobacco, pea, wheat, bean, and Swiss chard) and 3 without photorespiration (corn, sugarcane, and pigweed), were surveyed for peroxisomes. The distribution pattern for glycolate oxidase, glyoxylate reductase, catalase, and part of the malate dehydrogenase indicated that these enzymes exist together in this organelle. The peroxisomes were isolated at the interface between layers of 1.8 to 2.3 M sucrose by isopycnic nonlinear sucrose density gradient centrifugation or in 1.95 M sucrose on a linear gradient. Chloroplasts, located by chlorophyll, and mitochondria by cytochrome c oxidase, were in 1.3 to 1.8 M sucrose.In leaf homogenates from the first 7 species with photorespiration, glycolate oxidase activity ran,ged from 0.5 to 1.5 ,rmoles X min-' X g71 wet weight or a specific activity of 0.02 to 0.05 Atmole X min-' X mgV protein. Glyoxylate reductase activity was oomparable with glycolate oxidase. Catalase activity in the homogenates ranged from 4000 to 12,000 /moles X min-' X g-' wet weight or 90 to 300 ,umoles X min'1 X mg7 protein. Specific activities of malate dehydrogenase and cytochrome oxidase are also reported. In contrast, homogenates of corn and sugarcane leaves, without photorespiration, had 2 to 5 t% as much glycolate oxidase, glyoxylate reductase, and catalase activity. These amounts of activity, though lower than in plants with photorespiration, are, nevertheless, substantial. Peroxisomes were detected in leaf homogenates of all plants tested; however, significant yields were obtained only from the first 5 species mentioned above. From spinach and sunflower leaves, a maximum of about 50 %o of the marker enzyme activities was found to be in these microbodies after homogenization. The specific activity for peroxisomal glycolate oxidase and glyoxylate reductase was about 1 gmole X min-1 X mg-' protein; for catalase, 8000 tmoles X min-' X mg-1 protein,and for malate dehydrogenase, 40 ,mol.es X min-' X mg-' protein. Only small to trace amounts of marker enzymes for leaf peroxisomes were recovered on the sucrose gradients from the last 5 species of plants. Bean leaves, with photorespiration, had large amounts of these enzymes (0.57 umole of glycolate oxidase X min-' X g-1 tissue) in the soluble fraction, but only traces of activity in the peroxisomal fraction. Low peroxisome recovery from certain plants was attributed to particle fragility or loss of protein as well as to small numbers of particles in such plants as corn and sugarcane.Homogenates of pigweed leaves (no photorespiration) contained from one-third to one-half the activity of the glycolate pathway enzymes as found in comparable preparations from spinach leaves which exhibit photorespiration. However, only traces of peroxisomal enzymes were separated by sucrose gradient centrifugation of particles from pigweed. Data from pigweed on the absence of photorespiration yet abundance of enzymes associated with glycolate metabolism is inconsistent with current hypotheses ...
In cotyledons of sunflower seedlings glyoxysomal and peroxisomal enzymes exhibit different rates of development during germination. The total activity of isocitrate lyase, a glyoxysomal marker enzyme, rapidly increased during the first 3 days, and then decreased 89% by day 9. Exposure to light accelerated this decrease only slightly. The specific activity of glyoxysomal enzymes (malate synthetase, isocitrate lyase, citrate synthetase, and aconitase) in the microbody fraction from sucrose density gradients increased between days 2 and 4 about 2-to 3-fold, and thereafter it remained about constant in light or darkness.Total activity of the peroxisomal enzymes increased slowly in the dark during the first 4 days of germination and thereafter remained at a constant level of activity in the dark or increased 2-fold in 24 hours of light. The specific activties of glycolate oxidase, hydroxypyruvate reductase, and serine-glyoxylate aminotransferase in the isolated microbody fraction increased about 10-fold between days 2 and 4 in the dark and then remained constant or increased again 10-fold after an additional 48 hours in the light.The total activity of the common microbody marker, catalase, developed similarly to isocitrate lyase, but decreased only 72 % by day 9. The specific activities of enzymes (catalase, malate dehydrogenase, and aspartate aminotransferase) common to both microbody systems were 10-to 1000-fold greater than those of other enzymes. It is proposed that malate and aspartate may be involved in hydrogen transport between microbodies and other cellular sites.Glutamate-glyoxylate aminotransferase was very active in microbodies from castor bean endosperm and sunflower cotyledons. The specific activity of this aminotransferase developed similarly to glyoxysomal enzymes in the dark but further increased in the light, as did peroxisomal enzymes.The microbody fraction of castor bean endosperm germinated in the dark for 5 days contained both glyoxysomal and peroxisomal enzymes of similar specific activity.Adjacent to the microbody fraction on sucrose gradients from sunflower cotyledons were etioplasts at slightly lower densities and protein bodies at similar and higher densities. Their presence in the microbody fractions resulted in artificially low specific activities.
From spinach leaves (Xpinacea oleracea L.) two isoenzymes of glucosephosphate isomerase can be separated by DEAE-cellulose ion-exchange chromatography. Isoenzyme 1 eluting at high ionic strength shows a faster mobility during disc gel electrophoresis than isoenzyme 2 eluting at low ionic strength. For both isoenzymes the pH-optima and K,-values were very similar for either direction. However, the K,-values with glucose-6-phosphate as substrate (K, = 8.0 mM and 5.9 mM for isoenzyme 1 and 2, respectively) were 20-fold higher than with fructose-6-phosphate as substrate (K, = 300 pM for both isoenzymes). The molecular weight of both isoenzymes was estimated by sedimentation velocity centrifugation analysis to be 120000. The subcellular distribution reveals that isoenzyme 1 is located within the chloroplasts and isoenzyme 2 in the non-particulate cell fraction (cytosol).The results give further support to the view that in plants there exist two sets of isoenzymes for the oxidative pentose phosphate cycle, one set located in the chloroplasts, the other one in the non-particulate cell fraction (cyt,osoI [6]. The importance of the two sets of isoenzymes is to be seen in their intracellular location because one of each isoenzyme is located in the chloroplasts and the other one in the cytosol. Knowing that the chloroplast envelope represents a high barrier for the permeation of most sugar phosphates [7,8] the intracellular location ofAbbreviations. Glc-6-P, glucose 6-phosphate; Fru-6-P, fructose 6-phosphate.
Plastids from cotyledons of sunflower (Helianthus annus L.) seedlings, germinated in the dark or in the light, were isolated by isopycnic sucrose density gradient centrifugation. At all stages of development the whole plastids contained triose phosphate isomerase, NADPH-glyoxylate reductase, and l-dihydroxyphenylalanine oxidase, which were used as marker enzymes. At the beginning of germination the isopycnic density of whole plastids (proplastids) was about 1.22 g cm(-3). During development of proplastids into etioplasts in the dark, their isopycnic density increased to 1.26 g cm(-3). During exposure of germinating seedlings to white light for 2 days, the isopycnic density of whole plastids decreased from 1.26 to 1.22 g cm(-3). These changes in isopycnic density of plastids on sucrose density gradients are consistent with changes in the plastid ultrastructure caused by the protein-rich prolamellar body or by the lipid-rich thylakoids. Broken plastids (thylakoids), determined by the main peak of chlorophyll, increased in isopycnic density from less than 1.14 to about 1.17 g cm(-3) during illumination. During germination no major changes occurred in the isopycnic density of mitochondria. Microbodies had an isopycnic density of 1.24 g cm(-3) in very early stages of germination, and their density increased to 1.265 g cm(-3), when glyoxysomal enzymes reached maximum development.
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