Fumarase location

The method relies on an isotope effect so that H is removed in preference to 2H and 3H in the malate synthase reaction. In other words, the hydrogen isotope which is eliminated (in formation of the methylene group of malate) is determined by an intramolecular isotope effect such effects normally follow the sequence kH >k2H > k3H. Whether 3H is located in the pro-R or pro-S position of malate is then determined from the known selectivity of fumarase. 

Cell fractionation by mechanical rupture has already come under investigation. Two separate studies of mechanical rupture of yeast showed different rates of release for enzymes in different cell locations (13,14). Wall-linked and periplasmic enzymes were released relatively faster than total protein, soluble cytoplasmic enzymes at about the same rate, and the mitochondrial enzyme fumarase later than total protein (13). Proteolysis by the yeast s own enzymes was not found to be a problem. Activities of the released enzymes declined slowly or not at all when disruption was continued after the end of protein release, and the effect of shear was not separated from the effect of proteolysis. Shetty and Kinsella (15) also found a low rate of proteolysis after mechanical disruption, though thiol reagents added to weaken the cell walls before disruption caused an important increase in the extent of protein breakdown. 

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