Elucidating mechanism cellular uptake removal protein
In microorganisms, in which it is relatively easy to cause genetic mutations and to select specific mutants, this technique has been very useful.
In addition to their utility in the unraveling of metabolic pathways, the use of mutants in the early 1940s led to the postulation of the one gene-one enzyme hypothesis by the Nobel Prize winners Edward L.
Such measurements revealed the cyclic nature of the process; specific amino acids acted as catalysts, stimulating respiration to an extent greater than expected from the quantities added.
This was because the added material had been re-formed in the course of the cycle (Homogenates of tissue are useful in studying metabolic processes because permeability barriers that may prevent ready access of external materials to cell components are destroyed.
If fluoroacetic acid or fluorocitric acid is ingested by animals, for example, citric acid accumulates in the liver.
This correctly suggests that fluorocitric acid administered as such, or formed from fluoroacetic acid via the tricarboxylic acid (TCA) cycle, inhibits an enzyme of citrate oxidation.
Indeed, in the example, lactic acid is formed in response to abnormal circumstances and is not directly formed in the pathways of carbohydrate catabolism.
Second, the administration of metabolic poisons may lead to the accumulation of specific metabolites.
It had previously been believed that the proteins of tissues are stable once formed, disappearing only with the death of the cell.This procedure was used to show that isolated mitochondria catalyze the oxidation reactions of the TCA cycle and that these organelles also contain the enzymes of fatty acid oxidation.Similarly, isolated ribosomes are used to study the pathway and mechanism of protein synthesis.It is thus possible to obtain fractions containing predominantly one type of organelle: nuclei (and some unbroken cells); mitochondria, lysosomes, and microbodies; microsomes (i.e., ribosomes and endoplasmic reticulum fragments); and—after prolonged centrifugation at forces in excess of 100,000 times gravity—a clear liquid that represents the soluble fraction of the cytoplasm.The fractions thus obtained can be further purified and tested for their capacity to carry out a given metabolic step or steps.