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LDH Calcutta-1: a mutation of the B subunit of human lactate dehydrogenase
Insights
The human LDH Calcutta-1 variant, a form of lactate dehydrogenase, shows altered B-subunit structure. Homozygous variants exhibit reduced heat stability, suggesting a thermolabile B-subunit.
Area of Science:
- Biochemistry
- Human Genetics
- Enzymology
Background:
- The Calcutta-1 variant of human lactate dehydrogenase (LDH) is an electrophoretic variant found in India.
- LDH isoenzymes play crucial roles in cellular metabolism and are composed of different subunit combinations.
Purpose of the Study:
- To characterize the electrophoretic and heat stability properties of the human LDH Calcutta-1 variant.
- To investigate the molecular basis of the observed differences in the LDH Calcutta-1 variant.
Main Methods:
- Electrophoretic techniques including isoelectric focusing and denaturing gel electrophoresis.
- Heat stability assays on whole blood and purified LDH isoenzymes.
- Purification of the LD1 (B4) isoenzyme using affinity and ion-exchange chromatography.
Main Results:
- Isoelectric focusing revealed at least five Calcutta-1 LD1 bands.
- Denaturing gel electrophoresis showed two Calcutta-1 B subunit bands in variant samples, compared to one in normal samples.
- Homozygous Calcutta-1 LDH exhibited decreased heat stability, while heterozygous variant LDH showed normal heat stability.
Conclusions:
- The Calcutta-1 variant is associated with a B-subunit alteration affecting its electrophoretic mobility and heat stability.
- The thermolability is evident in the homozygous state, suggesting a structural modification of the B-subunit.
Abstract:
The electrophoretic variant of human LDH, Calcutta-1, occurs at phenotypic frequencies of 0-4% throughout India. The variant was examined by various electrophoretic techniques and by heat stability studies. The LD1 (B4) isoenzyme was purified from normal and variant bloods by affinity chromatography and ion-exchange chromatography. A minimum of five Calcutta-1 LD1 bands was demonstrated by isoelectric focusing. Electrophoresis of variant LD1 in high-molar urea-acrylamide denaturing gels resulted in two Calcutta-1 B subunit bands, while normal gels yielded only a single band. Homozygote Calcutta-1 LDH from red cells demonstrated a decreased heat stability, while heterozygote variant LDH showed a normal heat stability. This effect was confirmed when purified LD1's were compared. Evidence is presented suggesting a B-subunit variant showing thermolability in the homozygous form.