Mechanism of differential inhibition of lactate dehydrogenase isoenzymes in the BMC LD-1 assay.
The mechanism of inhibition of lactate dehydrogenase (LD) isoenzymes by guanidinium thiocyanate (GSCN) used in the LD-1 assay developed by Boehringer Mannheim Corporation (BMC) was investigated. Michaelis-Menten inhibition kinetics for the individual isoenzymes revealed that GSCN competitively inhibited LD-1 in the presence of lactate and NAD+, but is a noncompetitive inhibitor of LD-5. LD-2 and LD-3 exhibited mixed inhibition kinetics. The inhibition constants were two- to threefold smaller for LD-5 than for LD-1. Time-dependent studies also showed that the isoenzymes underwent a different rate of inactivation by GSCN. LD-5, LD-3, and LD-2 were rapidly inactivated within 1 min under the BMC assay conditions, whereas LD-1 lost only about 20% of activity after 10 min. The presence of lactate further protects LD-1, but not other isoenzymes. Under this condition, LD-1 was not inactivated during the initial 6 min of reaction. Separate experiments demonstrated that both guanidinium and thiocyanate ions are responsible for the inactivation process that was found to be irreversible. We speculate that GSCN selectively denatures the M subunit of LD. The H subunit is less susceptible to denaturation and is further stabilized by lactate.
Onigbinde
T A
TA
Department of Pathology and Laboratory Medicine, University of Texas Medical School, Houston 77030.
Wu
A H
AH
Wu
Y S
YS
Simmons
M J
MJ
Wong
S S
SS
D006146:Guanidines / Q000494:pharmacology*; D007527:Isoenzymes; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D007773:Lactates / Q000378:metabolism / Q000494:pharmacology; D019344:Lactic Acid; D009243:NAD / Q000378:metabolism / Q000494:pharmacology; D013861:Thiocyanates / Q000494:pharmacology*
D006146:Guanidines; D007527:Isoenzymes; D007773:Lactates; D013861:Thiocyanates; D009243:NAD; D019344:Lactic Acid; C054436:guanidine thiocyanate; D007770:L-Lactate Dehydrogenase