Peeter Sikk

Permeabilized cell and skinned fiber techniques in studies of mitochondrial function in vivo

In this chapter we describe in details the permeabilized cell and skinned fiber techniques and their applications for studies of mitochondrial function in vivo. The experience of more than 10 years of research in four countries is summarized. The use of saponin in very low concentration (50–100 μg/ml) for permeabilisation of the sarcolemma leaves all intracellular structures, including mitochondria, completely intact. The intactness of mitochondrial function in these skinned muscle fibers is demonstrated in this work by multiple methods, such as NADH and flavoprotein fluorescence studies, fluorescence imaging, confocal immunofluorescence microscopy and respiratory analysis. Permeabilized cell and skinned fiber techniques have several very significant advantages for studies of mitochondrial function, in comparison with the traditional methods of use of isolated mitochondria: (1) very small tissue samples are required; (2) all cellular population of mitochondria can be investigated; (3) most important, however, is that mitochondria are studied in their natural surrounding. The results of research by using this method show the existence of several new phenomenon — tissue dependence of the mechanism of regulation of mitochondrial respiration, and activation of respiration by selective proteolysis. These phenomena are explained by interaction of mitochondria with other cellular structures in vivo. The details of experimental studies with use of these techniques and problems of kinetic analysis of the results are discussed. Examples of large-scale clinical application of these methods are given. (Mol Cell Biochem 184: 81–100, 1998)

Hydrolysis of emulsified mixtures of triacylglycerols by pancreatic lipase

Hydrolysis of the emulsified mixture of short-chain triacylglycerols by porcine pancreatic lipase in the presence of procolipase and micellar sodium taurodeoxycholate has been studied. Increase in the content of tributyrin and trioctanoin in the mixture with triacetin had highly cooperative effects on the formation of the interfacial lipase procolipase complex. Abrupt enhancement of the complex stability was observed in the presence of 0.4-0.6 mol mol-1 of tributyrin or 0.58 mol mol-1 of trioctanoin in the substrate phase. The affinity of lipase towards interfacially bound procolipase for the trioctanoin containing 0.07-0.42 mol mol-1 of triacetin was approximately three times higher than that for pure trioctanoin. The cooperative processes involved in complex formation did not contribute to the affinity of the interfacial lipase/(pro)colipase complex towards substrate molecules and its catalytic activity.

Lipase-catalysed enantioselective hydrolysis: Interpretation of the kinetic results in terms of frontier orbital localisation

Abstract The initial velocities of the enantioselective hydrolysis of the esters derived from mono- and bicyclic alcohols and hemiacetals catalysed by Lipolase™ in water have been determined. The differences in hydrolysis rates within groups of sterically similar substrates have been interpreted in terms of frontier orbital localisation.

Enantioselective preparation of novel bicyclo[3.2.0]heptane derivatives using ester hydrolysis catalyzed by novo lipolaseTM

Abstract Double enantioselection occurs in the case of hydrolysis of (1S,2R,5S,6R)-2-bromo-3-butanoyloxy-6-hexanoyloxybicyclo[3.2.0]heptane (13), allowing us to obtain the corresponding bromodiol (−)-(14) and epoxyalcohol (−)-(15) of high optical purity.

High-purity porcine pancreatic lipase: novel effects in catalyzing hydrolysis of esters derived from bicyclo[3.2.0]hept-2-EN-6-OLS

Abstract The responsibility of high-purity porcine pancreatic lipase (PPL), an asymmetric catalyst, has been established quantitatively in the case of hydrolysis of the title compounds. A drastic differentiation between stereoisomeric as well as homologous substrates was observed.