Evidence for the lack of feedback regulation of gene activity and for the absence of subunit exchange between lactate dehydrogenase tetramers in vivo.

Abstract

Frog and rat lactate dehydrogenase (LDH, Llactate:NAD+ oxidoreductase, EC 1.1.1.27) isozymes, highly purified by affinity chromatography, were injected into newly fertilized frog (Rana pipiens) eggs to study the effect of the injected isozymes on the expression of the LDH genes of the de-veloping frog embryo. From these experiments three conclusions can be drawn: (i) homologous and heterologous LDH, even when present in more than twice the normal amount of LDH, does not play any role in-the switching on or off of the frog LDH genes or in regulating the level of gene activity. The intracellular levels of each LDH isozyme are dependent on both the rate constant for synthesis and the rate constant for degradation. It has been suggested that both constants are different for each isozyme and that the binomial distribution of the LDH isozymes is the result of a continuous in vivo assembly and disassembly of the LDH tetramers combined with a selective degradation of some of them (6-8). The intra-cellular occurrence of dissociation and reassociation of LDH tetramers has never been adequately tested.In the present investigation, these aspects of LDH regulation have been investigated by injecting homologous and heterologous LDH into newly fertilized Rana pipiens eggs. Three specific questions were examined: (a) Does MATERIAL AND METHODS Electrophoretic Separation. LDH isozyme separation was performed on starch gels, prepared as described by Boyer et al. (9). Resolution was achieved in 18 hr at 450 V. After electrophoresis was completed the gels were stained for LDH activity, as described by Markert and Faulhabr (10).Purification of LDH and Separation of the Isozymes. The LDH isozymes were purified from mouse and rat tissues by affinity chromatography with oxamate-aminohexyl-Sepharose-4B. LDH is bound to the immobilized analogue of pyruvate in the presence of NADH, and it is eluted when NADH is removed. This procedure gives a one step purification to homogeneity of all LDH isozymes studied (B. Nadal-Ginard, in preparation). The B4 isozymes were purified using a combination of blue dextran-Sepharose-4B (11) and oxamateaminohexyl-Sepharose-4B. LDH A4 was separated from the other isozymes on a DEAE-Sephadex A-50 column (12). After purification the LDH-isozymes were lyophilized and stored until used. LDH activity was measured as previously described (11).Rana pipiens Eggs. Mature eggs were obtained from gravid Rana pipiens females and fertilized according to procedures outlined in Rugh (13) as modified by Wright and Flathers (14). The eggs were "stripped" from the female into 100 mm Falcon petri dishes to form a circle around the edge of the dish and then covered with 10% Ringers solution. The jelly coat adheres strongly to the dish and immobilizes the egg during the injection procedure.Construction of Injection Pipettes. Coagulation glass capillary tubes (Fisher) were put on a vertical pipette puller (David Kopf Instruments) and the amount of heat in the coil regulated to produce a narrow bore section at least 8 mm...

Modulation of expression of LDH isoenzymes in endothelial cells by laminin: Implications for angiogenesis

“…The intracellular levels of each LDH isoenzyme are dependent on the rate of its synthesis and degradation. The LDH tetramer, and not the monomer, being the degradative unit, the distribution of the LDH isoenzyme is the result of continuous in vivo assembly and disassembly of LDH tetramers combined with the selective degradation of some of them [Nadal‐Ginard, 1976].…”

Developmental changes in translatable mRNAs for the cerebral enolase isozymes alphaalpha and gammagamma.

“…However, the proportion of adult a mRNA is three times higher than that of -y mRNA while the relative amounts of the corresponding antigens in adult rat brain , or mouse brain (PinScarna, 1980) are similar. This fact could be explained if the difference in the stability of these proteins observed in vitro Marangos et al, 1978) corresponds to different susceptibilities to degradation in vivo (Nadal-Ginard, 1976). Another explanation might be that a mRNA is translated more efficiently than Py mRNA in the reticulocyte lysate system.…”