Further Investigations on the Subunit Structure of Microsomal Carboxylesterases from Pig and Ox Livers

Abstract

Using sedimentation equilibrium studies, the molecular weight of pig liver esterase was found to be 163000. Sedimentation velocity experiments yielded a sedimentation coefficient s020,w of 7.9 S; the diffusion coefficient was found to be 40.7 μm2· s–1 by ultracentrifugation and 40.3 μm2· s–1 by analytical gel filtration. From these data a molecular weight of 181 000 was calculated. By analytical disc electrophoresis a molecular weight of 183000 was estimated. Disc electrophoresis of ox liver esterase revealed three enzymatically active bands. The molecular weight of two of them was 54500 and that of the third 187000. Extrapolation of the sedimentation coefficients measured at very low enzyme concentrations to infinite dilution yielded a minimum value of S020,w= 4.0 S. Based on gel filtration results a diffusion coefficient of 66.0 μm2· s–1 was calculated. From these data a molecular weight of 57300 was obtained. Sedimentation equilibrium experiments of the ox enzyme showed a non‐linear plot of In absorbance versus r2 indicating heterogeneity with respect to molecular weight. The smallest species present had a molecular weight of 59000. By sedimentation equilibrium experiments in the presence of 6 M guanidine‐HCl and 0.1 M 2‐mercaptoethanol, the subunit weight of pig liver esterase was found to be 58000. For the maleylated enzyme, values of 52500 (sedimentation equilibrium) and 56500 (from S20,w and D20,w) were obtained. By covalent cross linking of the subunits with dimethyl suberimidate and subsequent polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate, three bands of molecular weights 60000, 120000 and 180000 were found. All these results give strong evidence for a three subunit structure of pig liver carboxylesterase.

Chemical and Spectral Properties of Putidamonooxin, the Iron‐Containing and Acid‐Labile‐Sulfur‐Containing Monooxygenase of a 4‐Methoxybenzoate O‐Demethylase from Pseudomonas putida

“…Dimethyl suberimidate was prepared according to Davies and Stark [13]. Immunoglobin G was a gift from Dr Prenzel, University of Kiel, Germany, and covalently crosslinked pig liver esterase was prepared according to Junge et al [14]. All other chemicals and biochemicals were obtained from usual commercial sources, and were of the highest purity available.…”

“…55 + 3 0 (14) binding spectra decreases in going from 4-methoxybenzoate to benzoate with a concomitant increase in the K, values. Ks values taken from the substrate concentrations corresponding to the intersections of the experimental dependencies and the zero value of the function, shown in the inset of Fig.…”

An Enzymatic Chemical Amplifier Based on Mechanized Nanoparticles

“…The pore structure provides ample space for the encapsulation of the model enzyme (Porcine Liver Esterase), which has a hydrodynamic radius of about 4 nm. [22] …”

Studium der Mikroheterogenität und der Untereinheiten-Struktur von Carboxylesterasen durch quantitative Bestimmung der terminalen Aminosäuren

“…Bei der Rinderleberesterase beträgt das Untereinheiten-Gewicht ebenfalls ca. 60000 [10 > n J, jedoch ist hier die Neigung zur Assoziation wesentlich geringer^1 0 !. Um die Frage zu klären, ob die genannten trimeren Esterasen aus identischen oder verschiedenen Untereinheiten zusammengesetzt sind, erscheint eine quantitative Analyse der terminalen Aminosäuren als geeignetes Mittel.…”

“…Wir beschränk-ten unsere Untersuchung auf die Esterasen aus Schweineleber, Schweineniere und Rinderleber, deren Heterogenität von allen genannten Enzymen am wenigsten übersichtlich istt ? > 9 J. Wenn die quantitative Bestimmung der terminalen Aminosäuren mit genügender Genauigkeit ausgeführt werden kann, so sollte sich auch der durch hydrodynamische Verfahren erhaltene Wert für das Untereinheitengewichtl-2 ' 10 [19] . Alle übrigen Reagentien waren im Handel erhältlich.…”