Thermostability of lactate dehydrogenase LDH-A4 isoenzyme: Effect of heat shock protein DnaK on the enzyme activity

Cells exposed to temperature a few degrees higher than their growth temperature synthesize heat shock proteins (hsp) which may then compose even 20% of total protein content. This paper examined the in vitro protective effect of heat shock protein DnaK (70 kDa) from Escherichia coli against the heat inactivation of lactate dehydrogenase isoenzyme LDH-A₄. The LDH-A₄ isoenzyme was purified from fish skeletal muscle using the affinity chromatography on Oxamate-agarose. The enzyme was then heated in the absence and the presence of DnaK protein in a water bath at either 51 or 55°C. The LDH activity was determined by measuring the change in absorbency at 340 nm min⁻¹ at 30°C. The addition of DnaK protein to the LDH-A₄ isoenzyme before heat treatment can protect enzyme activity against mild thermal inactivation. Incubation of the LDH-A₄ isoenzyme at 51°C in the presence of DnaK protein stimulates its activity by about 30%. The presence of 2 mM ATP can raise LDH activity by another 10%. No significant recovery was observed when DnaK protein was added to LDH at 25°C following earlier inactivation. The maximal activities (V_max) in the presence of DnaK protein are almost twice those without DnaK protein in the case of heat-treated LDH-A₄ isoenzyme at 51°C. The observed protection of LDH-A₄ activity increased with the increasing DnaK protein concentration in the incubation medium. Results suggested that the presence of DnaK protein can protect LDH-A₄ from heat inactivation. This action may be important as a part of cellular chaperone machinery capable of repairing heat-induced protein damage. It may have a fundamental role in the acquisition of the thermotolerance to stress temperatures.