Unveiling Cell Ultrastructure

Classical microscopy was largely the occupation of professional cytologists. Some biochemists were excited by the possibilities of cytochemical staining for enzymes and the Feulgen technique for DNA, 

Preparation of biological material for electron microscopy still required fixation, dehydration, and ultrathin sections. Araldite and other resins were used in place of paraffin wax for blocking. At first, specially sharpened steel knives were employed to cut the sections, but from 1950 glass or diamond knives were used which could cut slices 100-200 nm thick. By 1952, Palade and others were obtaining sections 

Biochemical and ultrastructural analyses soon showed that the fractions obtained were inhomogeneous. Harvey (1931) had suggested 

Some of the results obtained by differential centrifugation showed enzyme distribution between different cell fractions which were difficult to interpret. Enzymes like carbamoyl phosphate synthase or isocitrate dehydrogenase were found both in mitochondria and in the soluble fraction of the cell. This led to detailed kinetic studies with purified enzymes which indicated there might be populations of enzymes with slightly different properties (isozymes) catalyzing similar reactions in different compartments or in different cell types. Variations in kinetic behavior appeared to tailor the enzyme appropriately to the particular compartment or cell where the reaction took place. 

Even greater uncertainty arose when only very small amounts of a constituent were detected outside the organelle in which it was principally concentrated. When the observation was reproducible, like the presence of small amounts of DNA ( 1% of the total cellular DNA) in highly purified mitochondria, it required considerable work on the size, composition, and organization of mitochondrial DNA before its significance was accepted. 

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