Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides is a reliable internal standard for radiation-inactivation studies of membranes in the frozen state.
The target size of four soluble enzymes (beta-galactosidase, pyruvate kinase, alcohol dehydrogenase, and glucose-6-phosphate dehydrogenase) in the presence or absence of subcellular membrane fractions has been determined by the radiation-inactivation method using samples in the frozen state. For each of the four enzymes, full activity was recovered after freezing and thawing in the absence of radiation. We found minimal (less than 20%) binding of the enzymes to either submitochondrial vesicles or sarcoplasmic reticulum vesicles. Under the conditions tested, beta-galactosidase, pyruvate kinase, and alcohol dehydrogenase exhibited target sizes which varied according to the experimental conditions, i.e., the buffer selected and also the presence or absence of membrane preparations. For these tetrameric enzymes, the target sizes were generally comparable to either a monomer or a dimer. By contrast, the target size of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides was found to be essentially invariant when frozen in a variety of buffers and in the presence or absence of either cryoprotectant (sucrose or glycerol) or different membrane preparations. The target size from 19 separate determinations gave an average value of 104 +/- 16 kDa, which is comparable to the molecular weight of the enzyme (104 kDa). We conclude that glucose-6-phosphate dehydrogenase from L. mesenteroides is a reliable internal standard for radiation-inactivation studies of membrane preparations in the frozen state.
D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000528:radiation effects*; D005615:Freezing; D005954:Glucosephosphate Dehydrogenase / Q000528:radiation effects; D007934:Leuconostoc / Q000201:enzymology*; D011770:Pyruvate Kinase / Q000528:radiation effects; D012015:Reference Standards; D012519:Sarcoplasmic Reticulum / Q000528:radiation effects; D001616:beta-Galactosidase / Q000528:radiation effects
D005954:Glucosephosphate Dehydrogenase; D011770:Pyruvate Kinase; D001616:beta-Galactosidase