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 degrees C. The LDH activity was determined by measuring the change in absorbency at 340 nm min-1 at 30 degrees 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 degrees 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 degrees C following earlier inactivation. The maximal activities (Vmax) in the presence of DnaK protein are almost twice those without DnaK protein in the case of heat-treated LDH-A4 isoenzyme at 51 degrees 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Zietara
M S
MS
Gdańsk University Biological Station, Gdańsk-Sobieszewo, Poland.
Skorkowski
E F
EF
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D004795:Enzyme Stability; D029968:Escherichia coli Proteins; D005399:Fishes; D018840:HSP70 Heat-Shock Proteins / Q000302:isolation & purification / Q000502:physiology*; D006358:Hot Temperature; D007527:Isoenzymes; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000201:enzymology*
D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D007527:Isoenzymes; D000255:Adenosine Triphosphate; D007770:L-Lactate Dehydrogenase; C063842:dnaK protein, E coli