Studies on the Aminotransferases Participating in the Glycolate Metabolism of the Alga Mougeotia

The intracellular distribution of enzymes capable of catalyzing the reactions from phosphoglycolate to glycerate in the bluegreen colored eucaryotic alga Cyanidium caldarium has been studied. After separating the organelles from a crude homogenate on a linear flotation gradient, the enzymes glycolate oxidase and glutamate-glyoxylate aminotransferase along with catalase were present in the peroxisomal fraction (density: 1.23 grams per cubic centimeter). Serine hydroxymethyltransferase was found in the mitochondrial fraction (density: 1.18 grams per cubic centimeter). In contrast to the observations in green leaves of higher plants, the enzymes for the conversion of serine to glycerate (serineglyoxylate aminotransferase and hydroxypyruvate reductase) were found only in the soluble fraction of the gradient. The partial characterization of enzymes from Cyanidium participating in glycolate metabolism revealed only slight differences from the corresponding enzymes from higher plants. The phylogenetic implications of the observed similarities between the enigmatic alga Cyanidium and higher plants are discussed.

Section: Serine-pyruvate Aminotransferasementioning

confidence: 99%

Subcellular Distribution of Enzymes of Glycolate Metabolism in the Alga Cyanidium caldarium

Groß

Beevers²

1989

“…However, there are a number of reports indicating that leaf extracts, or leaf peroxisomes, have substantial alanine:glyoxylate aminotransferase activity that may even exceed the glutamate:glyoxylate aminotransferase activity (4,7,21,24,25,28). Alanine is the main substrate for the formation of glycine also in the microbodies (peroxisomes) of the alga Mougeotia (29). In higher plants, most of the alanine:glyoxylate aminotransferase activity is attributed to the glutamate:glyoxylate aminotransferase (21,24,25).…”

mentioning

confidence: 99%

Aminotransfer from Alanine and Glutamate to Glycine and Serine during Photorespiration in Oat Leaves

Betsche

1983

ABSTRACT"N-Labeled glutamate and alanine were used to examine the photorespiratory nitrogen metabolism in oat (A vena sativa L.) leaf slices. Glutamate and alanine supply amino groups for glycine formation during photorespiration. The nitrogen flux from alanine to glycine was estimated to be 3 times higher than that from glutamate. It is concluded from these results that alanine is a direct and important amino donor for photorespiratory glycine formation in oat leaves. The 'N labeling of serine was almost as high as that of glycine during the initial period of the labeling experiments. Thereafter, the ratio of "N label in serine to "N label in glycine declined substantially.During photorespiration glycine is formed from glyoxylate. This process occurs in the leaf peroxisomes. The glycine enters the mitochondria, where two glycine are converted into one serine (24). In this complex reaction, CO2 and NH3 are liberated. The serine reenters the leaf peroxisomes, where serine:glyoxylate aminotransferase mediates the formation of hydroxypyruvate and glycine. By this process, one amino-nitrogen is directly recycled for glycine formation. The nitrogen of the other amino group, liberated as NH3, is refixed to glutamate via the glutamine synthetase/glutamate synthase pathway (1 1, 13, 22, 30). The finding that leaf peroxisomes contain glutamate:glyoxylate aminotransferase leads to the suggestion that the additional amino nitrogen needed for the formation of the second glycine is provided by this glutamate (13). However, there are a number of reports indicating that leaf extracts, or leaf peroxisomes, have substantial alanine:glyoxylate aminotransferase activity that may even exceed the glutamate:glyoxylate aminotransferase activity (4,7,21,24,25,28). Alanine is the main substrate for the formation of glycine also in the microbodies (peroxisomes) of the alga Mougeotia (29). In higher plants, most of the alanine:glyoxylate aminotransferase activity is attributed to the glutamate:glyoxylate aminotransferase (21,24,25). Glutamate:glyoxylate aminotransferase is also responsible for the glutamate:pyruvate and the alanine:a-ketoglutarate aminotransferase activities of leaf peroxisomes (3, 12,19,20 Preparation of Leaf Slices. The leaves were cut into slices (1.5-2.0 mm) with a razor blade. Four g of the leaf slices were vacuuminfiltrated in 40 ml of an osmotic medium consisting of 0.3 M sorbitol, 2 nim KH2PO4, 2 mM MgCl2, 1 mim MnCl2, 1 mm EDTA, and 5 mm Hepes/KOH, pH 7.9. Care was taken to release the vacuum slowly. Before use, the leaf slices were rinsed in a sieve with 40 ml of the same medium.Labeling Experiments. The leaf slices were transferred into a cuvette (8 x 5 x 2 cm) filled with 55 ml of the osmotic medium described above. The latter was bubbled with air for 3 h before being used. The leaf slices were stirred gently from the top, and the cuvette was illuminated with slide projectors from both sides the leaf slices were quickly transferred into a sieve, rinsed with 30 ml of water, and dipped several times into a large...

“…The activity of catalase in the test medium was inhibited by addition of 0.1 mM NaN3. All other enzymes were measured as already described: catalase, Cyt c oxidase, hydroxypyruvate reductase, and malate dehydrogenase (21); uricase (24); acyl-CoA dehydrogenase, enoyl-CoA hydratase, and hydroxyacyl-CoA dehydrogenase (28); acyl-CoA oxidase (8); glutamateglyoxylate aminotransferase and serinq-glyoxylate aminotransferase (30). The amino acids formed during the aminotransferase reactions were determined by HPLC analysis (10).…”

Section: Methodsmentioning

confidence: 99%

“…Their Microbodies have been demonstrated by electron microscopy to be present in many different algae (18), but only from a few of those organisms could the microbodies be isolated for biochemical characterization. Nevertheless, it has already become evident that there are different types ofalgal microbodies (7, 23).In Spirogyra and Mougeotia the peroxisomes are of the leaf type and are therefore involved in glycolate metabolism (22,25,30). On the other hand, the peroxisomes of the unicellular algae Chlorogonium, Chlamydomonas, ,Eremosphaera, and Polytomella contain neither enzymes of the glycolate metabolism nor enzymes ofthe glyoxylic acid cycle (6,20,21,24,27).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Characterization of Peroxisomes from the Alga Bumilleriopsis filiformis

Groß¹,

Winkler²,

Stabenau³

1985

The Xa"thophycean alp Bumilkropsis filiformis possesses peroxisomes which on electron micrographs show a mostly spherical or ovoid shape with a diameter in the range of 03 micrometer. Their Microbodies have been demonstrated by electron microscopy to be present in many different algae (18), but only from a few of those organisms could the microbodies be isolated for biochemical characterization. Nevertheless, it has already become evident that there are different types ofalgal microbodies (7, 23).In Spirogyra and Mougeotia the peroxisomes are of the leaf type and are therefore involved in glycolate metabolism (22,25,30). On the other hand, the peroxisomes of the unicellular algae Chlorogonium, Chlamydomonas, ,Eremosphaera, and Polytomella contain neither enzymes of the glycolate metabolism nor enzymes ofthe glyoxylic acid cycle (6,20,21,24,27). Thus, they have been regarded as unspecialized organelles.Until now algal microbodies of the glyoxysomal type have been isolated only from heterotrophically grown Euglena (1 1). Under autotrophic conditions, the corresponding organelles of this alga like leaf peroxisomes contain enzymes of glycolate metabolism (5). But these microbodies differ from leaf peroxisomes in that they contain a glycolate dehydrogenase rather than a glycolate oxidase (4, 16). Furthermore, there are also contrary reports whether catalase is a constituent of the organelles in Euglena (3,14,29).Recently it was reported that the unspecialized peroxisomes from Eremosphaera and the leaftype organelles from Mougeotia both contain enzymes of the #-oxidation pathway (28). Considering also the data for higher plants (8,9), it appeared that the possession of enzymes for the a-oxidation is a characteristic of ' Supported by the Deutsche Forschungsgemeinschaft.all different types of microbodies. This assumption now seems to be questionable since we have isolated peroxisomes from the Xanthophycean alga Bumilleriopsis which could not be demonstrated to contain enzymes for degradation of fatty acids. MATERIALS AND METHODSAlgal Material and Growth Conditions. Bumilleriopsisfiliformis (Xanthophyceae), strain 802-2, was obtained from the algae collection of the Institute for Plant Physiology, University of Gottingen, W. Germany.Cultures were grown autotrophically in glass tubes at 23YC in continuous light ofeither 3,000 or 20,000 lux in nutrient medium (12). The cultures were aerated with air plus 2% CO2 or air only.Preparation of Cell Homogenates and Separation of Organelles. Cells of 1 L of suspension were collected by low speed centrifugation and washed with grinding medium (28). For homogenization, the cells were transferred to 20 ml of the same medium containing 100 mg Polyclar AT, 20 mg cystein, 15 mg DTT, 70 mg BSA, and 15% (w/w) sucrose. Cells were broken in a Virtis homogenizer after adding 30 ml glass beads; the homogenate was centrifuged at 5OOg for 10 min. The resulting supernatant was used for sucrose density centrifugation (28).Assays. All assays for measuring the activities ofenzymes were ...