Isolation of Serine:Glyoxylate Aminotransferase from Cucumber Cotyledons

Abstract

Serine:glyoxylate aminotransferase, a marker enzyme for leaf peroxisomes, has been purified to homogeneity from cucumber cotyledons (Cucamis sativus cv Improved Long Green). The isolation procedure involved precipitation with polyethyleneimine, a two-step ammonium sulfate fractionation (35 to 45%), gel filtration on Ultrogel AcA 34, and ion exchange chromatography on diethylaminoethyl-cellulose, first in the presence of pyridoxal-5-phosphate, and then in its absence. The enzyme was purified approximately 690-fold to a final specific activity of 34.4 units per milligram. Electrophoresis of the purified enzyme on sodium dodecyl sulfate-polyacrylamide gels revealed two polypeptide bands with apparent molecular weights of approximately 47,000 and 45,000. Both polypeptides coeluted with enzyme activity under all chromatographic conditions investigated, both were localized to the peroxisome, and both accumulated in cotyledons as enzyme activity increased during development. The two polypeptides appear not to be structurally related, since they showed little immunological cross-reactivity and gave rise to different peptide fragments when subjected to partial proteolytic digestion. Antiserum raised against either the denatured enzyme or the 45,000-dalton polypeptide did not react with any other polypeptides present in a crude cotyledonary homogenate. The purified enzyme also had alanine:glyoxylate aminotransferase activity, but was about twice as active with serine as the amino donor.In cucurbits and related plant species with fat-storing cotyledons, the cotyledons serve as the site of lipid mobilization during early germination, then emerge above ground and become photosynthetic (2). The microbodies present at early stages (glyoxysomes) play a central role in fat mobilization, whereas those present after the onset of photosynthesis (peroxisomes) are involved in the glycolate pathway of photorespiration (2,28,29). The decrease in glyoxysomal enzyme activities usually occurs concomitantly with the increase in peroxisomal activities in the greening cotyledon (1,12,19,29). Much interest has focused on this changeover in microbody function (2,4,7,23,29), but the mechanism is still unresolved.

Purification and Characterization of an Anaerobically Induced Alanine Aminotransferase from Barley Roots

“…No activity was found when serine or aspartate were used as amino donors or when glyoxylate was used as a keto donor. Therefore, unlike several other aminotransferases that have been purified (10,12), this isoform is very specific in catalyzing the AlaAT reaction. A second distinctive feature of this enzyme is that PLP does not have any effect on either enzyme activity or column binding.…”

“…A number of different researchers have found that PLP affects aminotransferase activity or binding properties to chromatography columns (10,12). We incubated each pooled fraction containing AlaAT activity with 10 mm PLP; however, the presence of PLP had no effect on enzyme activity at any stage of purification.…”

“…Aminotransferases in plants have been shown to exhibit broad substrate specificities (10,12,19). We have assayed our purified AlaAT for the ability to catalyze a number of other transamination reactions.…”

“…Hatch identified two different AATs and three AlaATs in leaves of Atriplex spongiosa using ion-exchange chromatography (10). Hondred et al (12) identified a serine:glyoxylate aminotranferase from cucumber cotyledons and demonstrated that the pure enzyme had alanine:glyoxylate aminotransferase activity.…”

“…First, there are usually multiple isoforms of a specific aminotransferase within any plant tissue. Second, purified aminotransferases will usually use a variety of substrates in catalyzing a transamination reaction (10,12,26). Hatch identified two different AATs and three AlaATs in leaves of Atriplex spongiosa using ion-exchange chromatography (10).…”