We found that the UV spectrum of benzaldehyde semicarbazone shows a maximum at 280 nm instead of at 250 nm as shown by free aldehyde. The corresponding E(280) amounts to 21,000 instead of 13,000 at E(250) On this basis, we modified the spectrophotometric method of monoamine oxidase (MAO) estimation of Tabor and his colleagues. We measured the absorbancy increment at 280 nm after stopping the MAO catalyzed reaction with 0.77 mol/1 HC10(4), with 0.1 mol/1 semicarbazide hydrochloride (SC) added. Both time of incubation (1 h) and substrate concentration (3.33 x 10^-2 mol/1) were much greater than in the original method. SC inhibits MAO and it was omitted in the incubation medium. The method offers much greater sensitivity and can be used on large series of samples with homogenates and subcellular fractions as the source of enzyme.

A modification of Racker’s spectrophotometric method for the determination of fumarase (fumarate hydratase) and aconitase (aconitate hydratase) has been described. The described modification is more sensitive and more convenient than other methods. Beyond this, it may be used for the estimations in big series of samples. It also enables to obtain highest activities of both investigated enzymes at a suitable way of dilution, without the detergents. The inhibiting influence of DOC on fumarase but not on aconitase activity has been observed. Triton X-100 exerts no additional influence on the activity of both enzymes when compared with that of diluted samples without detergent.

Subfractionation of the purified preparations of mitochondria was performed according to Schnaitman’s digitonin method. In some experiments, polyvinyl sulphate (PVS) was added to the medium during the preparation and subfractionation of mitochondria. The formation of the ‘fluffy layer’ was not observed in the presence of PVS. The ‘fluffy layer’ was either removed or left within the pellet during the separation of the subfraction of mitoplasts from the supemate containing the outer membrane as well as the inter-membrane space. The monoamine oxidase (MAO) activity was determined by means of our own modification of Tabor’s method. In this way the influence of aldehyde oxidase upon the obtained results could be eliminated. A part of MAO activity was found in the subfraction of mitoplasts both in the presence and absence of PVS in the medium. The obtained results suggest double localization of MAO both in the outer and inner membrane. The influence of the method of determination of MAO activity on the evaluation of its intra-mitochondrial activity has been discussed.

The activation of 5'-nucleotidase in the homogenate as well as in the subcellular fractions, is due to the action of sodium deoxycholate and Triton X-100. Beyond this, both detergents cause a partial solubilization of the enzyme. The solubilized part of the enzyme is not activated during incubation with the detergents. The unsolubilized part of the enzyme is mainly, if not exclusively, activated. The existence of two forms of 5'-nucleotidase has been proposed on the basis of the above statements. These forms probably differ in their stability of binding to the membrane structure.

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