when assaying for LDH activity, how will you tell if your fractions contain LDH

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PURIFICATION OF LACTIC ACID DEHYDROGENASE (LDH) Mastery of a protein purification scheme is a rite of passage for all biochemists. The techniques employed in protein purification utilize important biochemical ideas about the structure, catalytic reactivity, and other characteristics of the protein of interest. We will perform a two step purification of LDH from chicken breast muscle. Lactic Acid Dehydrogenase in an important enzyme in the anaerobic metabolism of glucose for the generation of ATP. The activity of this enzyme is primarily responsible for explosive anaerobic athletic activity. The reaction that the enzyme catalyzes is the interconversion of pyruvate and lactate. Under anaerobic conditions, pyruvate is converted to lactate, with the concomitant conversion of NADH to NAD+. The regeneration of the NAD+ permits continued metabolic flux down the glycolytic pathway. This flow continues until the energy demands cease or until the cell and blood levels of lactate become intolerably high. Pyruvate + NADH ------------------> Lactic acid + NAD+ LDH LDH is a tetramer of 35 kd subunits. There are 2 types of subunits: and H form which predominates in the heart and an M form that predominates in the muscle and liver. These subunits can associate to form five types of tetramers (H4, H3L1, H2L2, H1L3, L4), all with LDH activity but with different substrate affinities and different responses to allosteric effectors. In general, enzymes that catalyze the same reaction but which differ in structure are referred to as isozymes or isoenzymes.

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