Carboxylesterase aus Rinderlebermikrosomen, I. Isolierung, Eigenschaften und Substratspezifität

Abstract

Zusammenfassung:1. Es wird über die Isolierung und Charakterisierung einer Carboxylesterase (EC 3.1.1.1) aus Rinderlebermikrosomen berichtet. Das Enzym wurde bei einer Ausbeute von 20% etwa TOfach angereichert (bezogen auf die spezifische Aktivität von Rinder lebermikr osomen).2. Die erhaltene Präparation erwies sich bei Säulenchromatographie, Gelfiltration und Papierelektrophorese als einheitlich. In der Hochspannungselektrophorese auf Stärkegel zeigte sich eine schneller wandernde, ebenfalls esteraseaktive Nebenbande. Untersuchungen zurSubstratspezifität ergaben, daß das Enzym eine Reihe von Carboxylestern und aromatischen Säureamiden hydrolysiert. Dabei wurden jedoch einige Unterschiede gegenüber den früher von uns isolierten Carboxylesterasen aus Leber und Niere des Schweines beobachtet. 4. Das Enzym katalysiert, neben der Hydrolyse zu Säure und Alkohol, auch die Übertragung von Aminosäureresten unter Bildung von Dipeptiden, wie z. B. von L-Phenylalanyl-L-Phenylalanin aus L-Phenylalanin-methylester und L-Tyrosyl-L-tyrosin aus L-Tyrosin-äthylester. 5. Das Enzym wird durch Organophosphat-Inhibitoren (E 600, Phosphorsäure-bis-[/?-nitro-phenylester]) hochempfindlich gehemmt. Summary:1. A carboxylesterase (EC 3.1.1.1) of beef liver microsomes was isolated and characterised. The enzyme was purified about 70-fold (based on the specific activity of the microsomes) in 20% yield.2. The preparation was homogeneous in column chromatography, gel-filtration and paper electrophoresis. In high voltage electrophoresis on starch gel, an additional faster-moving esterase-active band was detected.3. The enzyme hydrolyses a series of carboxyl esters and aromatic acid amides, but the substrate speci-ficity showed some differences from that of the carboxylesterases from the liver and kidney of the pig, which we described previously.4. Besides the hydrolysis into acid and alcohol, the enzyme also catalyses the transfer of amino acid residues to form dipeptides, e.g. L-phenylalanyl-Lphenylalanine from L-phenylalanine-methyl ester, and L-tyrosyl-L-tyrosine from L-tyrosine-ethyl ester.

Purification and Properties of Two Carboxylesterases from Rat‐Liver Microsomes

“…From the calculated values of the molecular weights it may easily be seen that the values are in agreement with a manomeric and a dimeric form of the enzyme. The presence of monomers and polymers of liver esterases has been discussed by Barker et al [42], Beniihr et al [43], and Runnegar et al [44]. By treatment with sodium dodecylsulphate, urea or guanidinium chloride, these authors were able to convert the enzyme from the dimer into the monomeric form.…”

“…I n the previous experiments the microsomal fraction contained about 70 ,IlO of the total homogenate activity. I n ox liver [12] and pig liver [39] the microsomes are reported to contain 65 -70°/, of the total esterase activity. I n the experiments presented in this work, only about 40°/, of the activity is found in the microsomal fraction while the nuclear fraction contained about the same activity.…”

“…These values are in the same order of magnitude as described earlier for pigand ox-liver esterases. Molecular weights of 80000 and 160000 [35], 174000 [41], 168000 [42] and 167000 [43] have been reported. From the calculated values of the molecular weights it may easily be seen that the values are in agreement with a manomeric and a dimeric form of the enzyme.…”

“…This result is in close agreement with values earlier reported 14,331, but differs slightly from the value 8. 4 given by Benohr et al [12]. The differences in these values are probably due to the substrates and buffers used in the determinations.…”

Carboxylesterases (EC 3.1.1). Purification and titration of ox liver carboxylesterase

“…W hile there are early reports on the partial purification of ox liver carboxylesterase (White, 1956;Kirkland, 1963), the first substantial purification was that of Benohr and Krisch (1967) whose method is similar to that reported earlier for the pig liver enzyme (Krisch, molecules, apparently distinguished by small charge differences.…”