Immunoisolation and the structural analysis of the sn-glycerol-3-phosphate dehydrogenase isozymes in mouse brain.

Isoenzymes of sn-glycerol-3-phosphate dehydrogen- ase (EC 1.1.1.8),which can be separated by ion ex- change chromatography, are differentially expressed during development.In the embryo and the neonatal brain the most abundant isozyme, the embryonic form, has the subunit composition EE and in the adult the most abundant isozyme, the adult form, has the subunit composition AA.A hybrid form AE can also be detected at most stages of development.In order to obtain com- parative protein structural information on these iso- zymes, we had to devise a novel procedure for isolating the embryonic isozyme because it was present at low concentrations.In this procedure: 1) AA isozyme was purified from skeletal muscle and antibody was produced against AA in rabbits; 2) since anti-AA antibody cross-reacted immunologically with AE but not EE, an immunoprecipitate, formed between anti-AA antibody and AE isozyme, was injected into a second rabbit which produced antibody that cross-reacted with all 3 isozymes; 3) a second immunoprecipitate made between anti-AE antibody and EE isozyme was injected into a third rabbit which produced antibody that re- acted well with AE and EE isozymes but poorly with AA.These antisera were used to immunoprecipitate partially purified `251-labeled isozymes.Electrophoresis of the precipitates in sodium dodecyl sulfate-polyacrylamide gels indicated that each subunit had identical apparent molecular weights of 37,000, indistinguisha- ble from a standard enzyme preparation stained with Coomassie blue.Cyanogen bromide fragmentation and limited proteolysis of the `25P-labeled protein did not reveal any unique peptides by one-dimensional electro- phoresis in sodium dodecyl sulfate-polyacrylamide gels.These results, coupled with evidence of immuno- logical cross-reactivity and reassociation of subunits in vitro to form hybrid molecular species, suggest that the isozymes are closely related in protein structure and that the embryonic isozyme does not represent a non- specific dehydrogenase activity.Embryos and certain undifferentiated tissues of mice ex- press a dimeric isozyme of sn-glycerol-3-phosphate dehydrogenase (EC 1.1.1.8)that can be distinguished biochemically from the major isozymic species found in adult tissues (1-3).These isozymes which are differentially expressed during de-