Synthesis and degradation of rat liver lactate dehydrogenase M4. Hybridization in the purification of lactate dehydrogenase isoenzymes.

For the measurement of lactate dehydrogenase (LDH) isoenzyme biosynthesis a new method is presented which is based on the quantitative hybridization of isoenzyme MI with isoenzyme Hq. After reversible dissociation the formation of active LDH M4 was more rapid and occurred to a greater extent than the formation of LDH Hq. The formation of hybrid isoenzymes from the two subunits resulted in an intermediate total activity. In the reaction mixture, 1 mM NADH resulted in a quantitative recovery of all isoenzymes. Rat liver proteins were labeled after a single injection of [3H]leucine or in double labeling experiments after injections of [Wlleucine and [3H]leucine. LDH Mr (LDH5) of the liver homogenate was separated from the other isoenzymes by DEAE-Sephadex chromatography. The LDH M4 fraction was subjected to a freeze-thaw cycle in neutral phosphate buffer containing 1 M NaCl and 1 lll~ NADH. A second DEAE-Sephadex chromatography was performed and the LDH M4 fraction obtained was hybridized with nonradioactive LDH H4 with the freeze-thaw technique. A third chromatography on DEAE-Sephadex was performed. The hybridized LDH M subunits attached to the column were eluted with a NaCl gradient. The radioactivity eluted was localized to the fractions containing LDH HMa, LDH H2M2, and LDH H3M. The radioactivity per unit of LDH M4 activity in the liver homogenate was calculated. Using this technique, it was demonstrated that cycloheximide, but not actinomycin D, inhibited the incorporation of radioactive leucine into LDH M+ This indicates that the synthesis of lactate dehydrogenase is coded by a relatively stable messenger RNA. The half-life of rat liver LDH M4 was found to be 4.1 days. The degradation constant, kd, was 0.173 day-‘. The synthesis rate, k,, was calculated to be 84 molecules. cell-l. s-l.