The Journal of Cell Biology, Vol 88, 492-498, Copyright © 1981 by The Rockefeller University Press
Regulation of the synthesis of lactate dehydrogenase-X during spermatogenesis in the mouse
ED Wieben
Total mouse testis RNA directs the synthesis of the sperm-specific C
subunit of lactate dehydrogenase-X (LDH-X) when translated in a cell- free
system derived from rabbit reticulocytes. The newly synthesized C subunits
were isolated by immunoprecipitation with antibody specific for this
isozyme, and quantitated by electrophoresis on SDS polyacrylamide gels. The
amount of radioactivity incorporated into the enzyme subunit was directly
proportional to the amount of testis RNA added to the translational system,
thereby providing a sensitive and reliable method for assessing relative
LDH-X mRNA activity. A combination of sucrose gradient centrifugation and
oligo(dT)-cellulose chromatography resulted in a 23-fold purification of
LDH-X mRNA over total cytoplasmic testis RNA. Analysis of LDH-X mRNA
activity in the developing testis indicated that the appearance of
functional LDH-X mRNA activity coincides with the appearance of LDH-X
catalytic activity at 14 d postpartum. Measurement of LDH-X mRNA levels in
separated testis cell populations prepared by centrifugal elutriation
demonstrated that LDH-X mRNA represents 0.17-0.18% of the total functional
mRNA activity in fractions enriched in pachytene spermatocytes and round
spermatids, but only 0.09-0.10% of the translation products of elongated
spermatids.
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