ABSTRACT
1. We have quantified lactate dehydrogenase (LDH) A, B and C enzyme levels during the first twelve weeks of life in rat testes. 2. Proteins were electrophoresed under non-denaturing conditions, isoenzymes identified by activity staining and quantified by densitometry scanning. 3. LDH A declined 2-3-fold during testes growth, changing from the most to the least abundant isoenzyme form. LDH B remained fairly constant, and LDH C increased from undetectable levels at birth to become the most abundant of the three polypeptides from week 3 onwards.
Subject(s)
L-Lactate Dehydrogenase/metabolism , Testis/growth & development , Aging/metabolism , Animals , Isoenzymes , Macromolecular Substances , Male , Rats , Rats, Inbred Strains , Testis/enzymologyABSTRACT
RNA was isolated from rat liver and heart tissues at various times up to 12 weeks after birth, and probed on slot blots with lactate dehydrogenase A and B cDNA probes. Although the relative abundances of LDH A in liver and LDH B in heart increased substantially in the 12 weeks after birth, mRNAs for both isoenzymes remained remarkably stable in both tissues over the same period. The implications of these observations for the regulation of constitutive gene expression are discussed.
Subject(s)
L-Lactate Dehydrogenase/metabolism , Liver/enzymology , Myocardium/enzymology , RNA, Messenger/metabolism , Aging/metabolism , Animals , Animals, Newborn/genetics , Blotting, Northern , DNA Probes , Gene Expression Regulation, Enzymologic , L-Lactate Dehydrogenase/genetics , Macromolecular Substances , Rats , Rats, Inbred StrainsABSTRACT
Inclusion of aurintricarboxylic acid (ATA) in extraction buffers for the isolation of RNA from animal tissues resulted in high yields (0.5-2.0 mg/g of tissue) of undegraded material as judged by agarose-gel-electrophoretic analyses and Northern-blotting experiments. However, ATA bound to nucleic acids, forming stable complexes, and so we have established methods for spectrophotometric quantification of RNA in these coloured complexes, and for easy removal of sufficient ATA to leave RNA in a consistently hybridizable condition at the end of a purification. The use and subsequent removal of ATA was straightforward and gave satisfactory results for all rat tissues tested.